Glycogen Synthase Kinase 3

GLYCOGEN SYNTHASE KINASE 3 (GSK-3) AND ITS INHIBITORS WILEY SERIES IN DRUG DISCOVERY AND DEVELOPMENT Binghe Wang, Ser...

Author: Ana Martinez | Ana Castro | Miguel Medina | Binghe Wang

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GLYCOGEN SYNTHASE KINASE 3 (GSK-3) AND ITS INHIBITORS

WILEY SERIES IN DRUG DISCOVERY AND DEVELOPMENT Binghe Wang, Series Editor

Computer Applications in Pharmaceutical Research and Development Edited by Sean Ekins Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors: Drug Discovery and Development Edited by Ana Martinez, Ana Castro, and Miguel Medina

GLYCOGEN SYNTHASE KINASE 3 (GSK-3) AND ITS INHIBITORS Drug Discovery and Development

EDITED BY

ANA MARTINEZ ANA CASTRO MIGUEL MEDINA

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2006 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and speciﬁcally disclaim any implied warranties of merchantability or ﬁtness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of proﬁt or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: Glycogen synthase kinase 3 (GSK-3) and its inhibitors : drug discovery and development / [edited by] Ana Martinez, Ana Castro, Miguel Medina. p. cm. – (Wiley series in drug discovery and development) Includes index. ISBN-13: 978-0-471-77001-5 (cloth) ISBN-10: 0-471-77001-9 (cloth) 1. Glycogen synthase kinase-3. 2. Glycogen synthase kinase-3 – Inhibitors. I. Martínez, Ana, 1961– . II. Castro, Ana. III. Medina, Miguel, 1959– . IV. Series. [DNLM: 1. Protein Kinase Inhibitors–therapeutic use. 2. Glycogen Synthase Kinase 3–antagonists & inhibitors. 3. Glycogen Synthase Kinase 3–physiology. QU 143 G568 2006] QP606.P76G59 2006 2006007195 572´.567–dc22

Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1

To our families and co-workers that hold and help us in our research

CONTENTS

FOREWORD—THE ORIGINS OF GSK-3 PREFACE

ix xvii

ACKNOWLEDGMENTS

xix

CONTRIBUTORS

xxi

ABBREVIATIONS

xxv

PART I

CELLULAR FUNCTIONS OF GSK-3

1

1

Glycogen Synthase Kinase 3: An Introductory Synopsis

2

Glycogen Synthase Kinase 3b (GSK-3b) a Key Signaling Enzyme: A Developmental Neurobiological Perspective

25

Role of GSK-3/Shaggy in Neuronal Cell Biology

45

3

4 The Crystal Structures of Glycogen Synthase Kinase 3 5

Kinase–Kinase and Site–Site Interactions in the Phosphorylation of Tau by GSK-3

3

61

83 vii

viii

CONTENTS

PART II

GSK-3 AS A THERAPEUTIC TARGET IN HUMAN PATHOLOGY

6 GSK-3, a Key Player in Alzheimer’s Disease 7

105 107

Glycogen Synthase Kinase 3: A Target for Novel Mood Disorder Treatments

125

8

GSK-3 and Stem Cells

155

9

Glycogen Synthase Kinase 3: Role in Neurodegeneration and Neuroprotection

173

10

Protein Kinase Assays for Drug Discovery

189

11

Animal Models with Modiﬁed Expression of GSK-3 for the Study of Its Physiology and of Its Implications in Human Pathologies

203

PART III

GSK-3 INHIBITORS: DEVELOPMENT AND THERAPEUTIC POTENTIAL

221

12

Lithium, the Seminal GSK-3 Inhibitor

223

13

Inhibition of GSK-3 as Therapeutic Strategy in Disease: Efﬁcacy of AR-A014418

243

14 TDZD’s: Selective and ATP Noncompetitive Glycogen Synthase Kinase 3 Inhibitors 15

16

257

3-Amino Pyrazoles as Potent and Selective Glycogen Kinase Synthase 3 (GSK-3) Inhibitors

281

Marine Compounds as a New Source for Glycogen Synthase Kinase 3 Inhibitors

307

INDEX

333

FOREWORD—THE ORIGINS OF GSK-3

THE DISCOVERY OF GSK-3 It is a great honour to have been asked to write the introduction to this book, which celebrates 25 years of research on glycogen synthase kinase 3 (GSK-3), and to recall the events that led to the identiﬁcation and characterisation of this fascinating protein kinase, which is involved in regulating so many cellular processes. In late 1971 I had joined the Department of Biochemistry at the University of Dundee as a junior member of the Faculty after spending two years as a postdoctoral fellow studying the regulation of glycogen metabolism in Edmond’s Fischer’s laboratory at the University of Washington, Seattle. At that time only four enzymes were known that were regulated by phosphorylation, namely glycogen phosphorylase (activated by phosphorylase kinase) [1], phosphorylase kinase [2] and glycogen synthase [3] (activated and inactivated, respectively, by cyclic AMP-dependent protein kinase, PKA), and pyruvate dehydrogenase (inactivated by pyruvate dehydrogenase kinase) [4]. No protein kinase had been puriﬁed to homogeneity or characterised in molecular terms, and so I decided to tackle phosphorylase kinase. This led me to discover that its regulation by PKA involved its phosphorylation at two serine residues, one on the α-subunit and one on the β-subunit of the enzyme. This was unexpected, because the regulation of glycogen phosphorylase involved the phosphorylation of just one serine residue, and I was intrigued to know whether this was a common phenomenon. Looking for another example of “multisite” phosphorylation, I could see from the literature that glycogen synthase was a potential candidate and, armed with a grant from The British Diabetic Association (today called Diabetes UK), I was able to appoint my ﬁ rst postdoctoral fellow, Hugh Nimmo, to start work on this problem in late 1973. ix

x

FOREWORD—THE ORIGINS OF GSK-3

Quite highly puriﬁed preparations of muscle glycogen synthase were always contaminated with traces of PKA, so that glycogen synthase became phosphorylated if it was incubated with MgATP. In order to suppress this activity Hugh used a potent and speciﬁc protein inhibitor of PKA, termed the “protein kinase inhibitor” (PKI) but, to his surprise, PKI only partially suppressed the phosphorylation of glycogen synthase. Moreover, the residual glycogen synthase kinase activity that remained even in the presence of PKI could not be PKA, because it phosphorylated glycogen synthase at a site(s) that could not be rendered soluble in trichloroacetic acid by digestion with trypsin. In contrast, the major site(s) phosphorylated by PKA was (were) solubilised by trypsin [5]. These experiments demonstrated that puriﬁed preparations of glycogen synthase were contaminated with another protein kinase(s), which we termed glycogen synthase kinase 2 (GSK-2) to distinguish it from PKA (GSK-1). The next step was to purify GSK-2, but it soon became clear that PKA and GSK-2 were not the only glycogen synthase kinases in muscle. One of these protein kinases was not associated with the glycogen particles to which glycogen synthase was attached and was provisionally called GSK-3 in the Colworth Medal Lecture of the Biochemical Society that I delivered in July 1978 [6]. The partial puriﬁcation and characterisation of GSK-3 was carried out by Noor Embi, a graduate student from Malaysia (currently the Head of the Malaysian Biotechnology Directorate), while an Australian postdoc Dennis Rylatt identiﬁed three of the serine residues on glycogen synthase that are targeted by GSK-3, and found that they were all located in the same tryptic peptide. These papers were submitted for publication at the end of 1979 and published in the European Journal of Biochemistry in 1980 [7,8]. A postdoc Brian Hemmings [9] and then graduate student Jim Woodgett [10] later completed the puriﬁcation of GSK-3 to homogeneity. Jim has subsequently become one of leading researchers on GSK-3 and he has contributed the ﬁ rst chapter of this book.

INSULIN AND GSK-3 In 1960 Joseph Larner reported that the activity of glycogen synthase was increased within minutes when rat diaphragms were incubated with insulin [11] and, a few years later, he showed that this resulted from decreased phosphorylation of this enzyme [12]. Following the discovery that PKA can phosphorylate and inhibit GSK-3 in vitro [13,14], it was thought that insulin must exert its effect on glycogen synthase by inhibiting PKA, but no decrease in the concentration of cyclic AMP could be detected in muscle under conditions where insulin stimulated glycogen synthase [12]. This led Joe Larner to suggest that insulin might trigger the formation of a “second messenger” or chemical mediator” distinct from cyclic AMP, which bound to PKA and prevented its activation by cyclic AMP but, despite much effort, no such molecule

FOREWORD—THE ORIGINS OF GSK-3

xi

was ever puriﬁed and characterised. However, when other glycogen synthase kinases were identiﬁeid, I suggested that insulin might instead activate glycogen synthase by inhibiting GSK-2 [5] and/or GSK-3, rather than PKA [6]. Strong support for this new idea came in 1983 when Peter Parker, a postdoc in my lab, demonstrated that the major residues on glycogen synthase that underwent dephosphorylation in response to insulin were those targeted by GSK-3, and not those targeted by PKA or other protein kinases [15]. In the early 1990’s Jim Woodgett and Bill Benjamin [16], and another former graduate student Chris Proud [17], were able to establish that GSK-3 was indeed inhibited within minutes when cells were stimulated with insulin. My student Darren Cross then showed that the insulin-induced inhibition of GSK-3 was prevented by inhibition of phosphatidylinositol (PI) 3-kinase and reversed by treatment with a serine/threonine-speciﬁc phosphatase [18], which demonstrated that the inhibition of GSK-3 resulted from its phosphorylation of a serine or threonine residue. The relevant residues were identiﬁed as Ser21 in GSK-3α and Ser9 in GSK-3β and the insulin stimulated protein kinase that inactivated GSK-3 was identiﬁed by Darren, as protein kinase B (PKB, also called Akt) in a collaboration with Brian Hemmings [19]. Dario Alessi, a postdoc, found that the activation of PKB was mediated by its phosphorylation [20] and he went on to identify and characterise a protein kinase that phosphorylated and activated PKB, but only in the presence of lipid vesicles containing PI(3,4,5)trisphosphate, the product of the PI 3-kinase reaction [21]. We therefore termed this enzyme 3-phosphoinositidedependent protein kinase 1 (PDK1). The discovery of PDK1 closed the chain of events by which insulin inhibits GSK-3 and made it possible to write a complete outline of this signalling pathway. The pathway was later validated genetically [22] and, in particular, Dario’s group showed that insulin was unable to activate muscle glycogen synthase in mice that express knock-in mutants of GSK-3 that cannot be inactivated by PKB [23].

THE UNUSUAL SUBSTRATE SPECIFICITY OF GSK-3 In the early 1980’s I found that glycogen synthase was phosphorylated constitutively by a protein kinase that I initially termed GSK-5, but which is nowdays called CK2. However, in contrast to GSK-3, the phosphorylation of glycogen synthase by CK2 did not decrease its activity [24]. Colin Picton, a postdoc in the lab, then made the surprising ﬁ nding that, although phosphorylation by CK2 did not alter the activity of glycogen synthase directly, it was a prerequisite for the GSK-3-catalysed phosphorylation and inactivation of glycogen synthase [25]. Colin also identiﬁed the serine residue phosphorylated by CK2 and showed that it was located just C-terminal to the serine residues phosphorylated by GSK-3 [26]. The function of CK2-catalysed phosphorylation was therefore to “prime” glycogen synthase for phosphorylation

xii

FOREWORD—THE ORIGINS OF GSK-3

by GSK-3. Brian Hemmings in the lab then found that phosphorylation by CK2 also “primed” the Type-2 regulatory subunit of cyclic AMP-dependent protein kinase for phosphorylation by GSK-3 [27], demonstrating that the requirement for a “priming phosphorylation” is not unique to glycogen synthase. These observations were extended by Peter Roach in elegant experiments which demonstrated that the optimal recognition sequence for GSK-3 is Ser/Thr-Xaa-Xaa-Xaa-pSer/pThr (where pSer and pThr are phosphoserine and phosphothreonine, respectively, and Xaa is any amino acid) [28]. We now know that the prior action of a “priming” kinase is critical for the GSK-3catalysed phosphorylation of many proteins and that the identity of the “priming”kinase varies from substrate to substrate. Much later, the speciﬁc binding site for the priming phosphate of the substrate was identiﬁed, and the PKB-catalysed phosphorylation of GSK-3 was shown to transform its N-terminus into a pseudosubstrate which inhibits GSK-3 by interacting with the binding site for the priming phosphate [29,30]. It is therefore important to emphasise that the extent to which phosphorylation of GSK-3 inhibits activity is likely to vary from substrate to substrate and will clearly depend on the strength of interaction between GSK-3 and its different substrates. Phosphorylation may therefore only inhibit the phosphorylation of a subset of the substrates of GSK-3 under physiological conditions, a critical point that is frequently overlooked.

GSK-3: THE PRESENT AND THE FUTURE Although GSK-3 was originally identiﬁed as a protein kinase involved in the regulation of glycogen metabolism, we now know that it participates in the control of many cellular processes, including embryonic development, where it is a key component of the Wnt signalling pathway, as well as gene transcription and neuronal cell function, which will be apparent from reading the chapters in this book. Undoubtedly, many more substrates for GSK-3 remain to be discovered in these and other processes. Another extremely exciting development in recent years has been has the advent of potent and speciﬁc inhibitors of GSK-3, which have not only become powerful pharmacological reagents with which to study its functions, but also have therapeutical potential for the treatment of diabetes, stroke, Alzheimer’s and other diseases. These aspects are the major topic of Sections B and C of this book. However, these compounds are still at the preclinical stage and whether inhibitors of GSK-3 can be used safely for prolonged periods remains to be seen. While I was writing this foreword I noticed that well over 1000 papers were published over the two year period from January 1st 2004 to December 31st 2005, many more than in the previous two years. Thus research on GSK-3 is clearly continuing at an ever increasing pace and the study of this enzyme is clearly going to continue to be a vibrant and active ﬁeld of research for many years to come.

FOREWORD—THE ORIGINS OF GSK-3

xiii

REFERENCES 1. 2.

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Krebs E.G., Fischer E.H. (1956). The phosphorylase b to a converting enzyme of rabbit skeletal muscle. Biochim. Biophys. Acta., 20, 150–157. DeLange R.J., Kemp R.G., Riley W.D., Cooper R.A., Krebs E.G. (1968). Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3′,5′-monophosphate. J. Biol. Chem., 243, 2200–2208. Friedman D.L., Larner J. (1963). Studies on UDPG-Alpha-Glucan Transglucosylase. Iii. Interconversion of Two Forms of Muscle UDPG-Alpha-Glucan Transglucosylase by a Phosphorylation-Dephosphorylation Reaction Sequence. Biochemistry., 2, 669–675. Linn T.C., Pettit F.H., Reed L.J. (1969). Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc. Natl. Acad. Sci. U. S. A., 62, 234–241. Nimmo H.G., Cohen P. (1974). Glycogen synthetase kinase 2 (GSK-2); the identiﬁcation of a new protein kinase in skeletal muscle. FEBS Lett., 47, 162– 166. Cohen P. (1979). The hormonal control of glycogen metabolism in mammalian muscle by multivalent phosphorylation. Biochem. Soc. Trans., 7, 459–480. Embi N., Rylatt D.B., Cohen P. (1980). Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem., 107, 519–527. Rylatt D.B., Aitken A., Bilham T., Condon G.D., Embi N., Cohen P. (1980). Glycogen synthase from rabbit skeletal muscle. Amino acid sequence at the sites phosphorylated by glycogen synthase kinase-3, and extension of the N-terminal sequence containing the site phosphorylated by phosphorylase kinase. Eur. J. Biochem., 107, 529–537. Hemmings B.A., Yellowlees D., Kernohan J.C., Cohen P. (1981). Puriﬁcation of glycogen synthase kinase 3 from rabbit skeletal muscle. Copuriﬁcation with the activating factor (FA) of the (Mg-ATP) dependent protein phosphatase. Eur. J. Biochem., 119, 443–451. Woodgett J.R., Cohen P. (1984). Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate speciﬁcity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5). Biochim. Biophys. Acta., 788, 339– 347. Villar-Palasi C., Larner J. (1960). Insulin-mediated effect on the activity of UDPG-glycogen transglucosylase of muscle. Biochim. Biophys. Acta., 39, 171– 173. Craig J.W., Larner J. (1964). Inﬂuence of Epinephrine and Insulin on Uridine Diphosphate Glucose-Alpha-Glucan Transferase and Phosphorylase in Muscle. Nature., 202, 971–973. Bishop J.S., Larner J. (1969). Presence in liver of a 3′:5′-cyclic AMP stimulated kinase for the I form of UDPG-glycogen glucosyltransferase. Biochim. Biophys. Acta., 171, 374–377. Soderling T.R., Hickenbottom J.P., Reimann E.M., Hunkeler F.L., Walsh D.A., Krebs E.G. (1970). Inactivation of glycogen synthetase and activation of

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phosphorylase kinase by muscle adenosine 3′,5′-monophosphate-dependent protein kinases. J. Biol. Chem., 245, 6317–6328. 15. Parker P.J., Caudwell F.B., Cohen P. (1983). Glycogen synthase from rabbit skeletal muscle; effect of insulin on the state of phosphorylation of the seven phosphoserine residues in vivo. Eur. J. Biochem., 130, 227–234. 16. Hughes K., Ramakrishna S., Benjamin W.B., Woodgett J.R. (1992). Identiﬁcation of multifunctional ATP-citrate lyase kinase as the alpha-isoform of glycogen synthase kinase-3. Biochem. J., 288 (Pt 1), 309–314. 17. Welsh G.I., Proud C.G. (1993). Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B. Biochem. J., 294 (Pt 3), 625–629. 18. Cross D.A., Alessi D.R., Vandenheede J.R., McDowell H.E., Hundal H.S., Cohen P. (1994). The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem. J., 303 (Pt 1), 21–26. 19. Cross D.A., Alessi D.R., Cohen P., Andjelkovich M., Hemmings B.A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature., 378, 785–789. 20. Alessi D.R., Andjelkovic M., Caudwell B., Cron P., Morrice N., Cohen P., Hemmings B.A. (1996). Mechanism of activation of protein kinase B by insulin and IGF-1. Embo. J., 15, 6541–6551. 21. Alessi D.R., James S.R., Downes C.P., Holmes A.B., Gaffney P.R., Reese C.B., Cohen P. (1997). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol., 7, 261–269. 22. Mora A., Komander D., van Aalten D.M., Alessi D.R. (2004). PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell. Dev. Biol., 15, 161–170. 23. McManus E.J., Sakamoto K., Armit L.J., Ronaldson L., Shpiro N., Marquez R., Alessi D.R. (2005). Role that phosphorylation of GSK-3 plays in insulin and Wnt signalling deﬁ ned by knockin analysis. Embo. J., 24, 1571–1583. 24. Cohen P., Yellowlees D., Aitken A., Donella-Deana A., Hemmings B.A., Parker P.J. (1982). Separation and characterisation of glycogen synthase kinase 3, glycogen synthase kinase 4 and glycogen synthase kinase 5 from rabbit skeletal muscle. Eur. J. Biochem., 124, 21–35. 25. Picton C., Woodgett J., Hemmings B., Cohen P. (1982). Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3. FEBS Lett., 150, 191–196. 26. Picton C., Aitken A., Bilham T., Cohen P. (1982). Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. Eur. J. Biochem., 124, 37–45. 27. Hemmings B.A., Aitken A., Cohen P., Rymond M., Hofmann F. (1982). Phosphorylation of the type-II regulatory subunit of cyclic-AMP-dependent

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protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase 5. Eur. J. Biochem., 127, 473–481. 28. Fiol C.J., Mahrenholz A.M., Wang Y., Roeske R.W., Roach P.J. (1987). Formation of protein kinase recognition sites by covalent modiﬁcation of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J. Biol. Chem., 262, 14042–14048. 29. Frame S., Cohen P., Biondi R.M. (2001). A common phosphate binding site explains the unique substrate speciﬁcity of GSK-3 and its inactivation by phosphorylation. Mol. Cell., 7, 1321–1327. 30. Dajani R., Fraser E., Roe S.M., Young N., Good V., Dale T.C., Pearl L.H. (2001). Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate speciﬁcity and autoinhibition. Cell., 105, 721–732.

PREFACE

Glycogen synthase kinase 3 (GSK-3) is an “old enzyme” discovered about 20 years ago. However, there is a renewed interested in this enzyme in the twenty-ﬁ rst century because of the very promising potential of its inhibitors for the treatment of several diseases as neurodegenerative disorders (Alzheimer’s disease), type 2 diabetes, depression and bipolar disorders, stroke, acute inﬂammatory processes, cancer, and so forth. Consequently, GSK-3 is considered as one of the most promising drug targets. Along this line, recent years have witnessed the birth of several chemical families of GSK-3 inhibitors with varying selectivity. These compounds have been extensively tested, and shown great potential for treatment of diseases that have no current effective treatment. Gathered and systematically analyzed in this volume is the relevant up-to-date information concerning GSK-3 and its known inhibitors. This volume should be useful to researchers interested in drug design and development, especially those researchers directly involved in the fascinating world of GSK-3. Ana Martinez, Ana Castro, and Miguel Medina

xvii

ACKNOWLEDGMENTS

The launch of a research volume is a multifaceted task that can only be acomplished by team work. Our ﬁ rst and foremost acknowledgment has to go to Constanza Niell for her invaluable assistance in the preparation of this volume. We are additionally very grateful to all the people who contributed to this book. Last, we wish to thank our many colleagues and collaborators, including our former and present students and post-docs, from whom we have learned so much over the years.

xix

CONTRIBUTORS

Alonso, Diana, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Alonso, Mercedes, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Avila, Jesús, Centro de Biología Molecular “Severo Ochoa” CSIC/UAM. Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Bhat, Ratan, AstraZeneca R&D, Södertälje, Disease Biology, SWEDEN 151 85 Budd Haeberlein, Samantha L., AstraZeneca R&D, Södertälje, Disease Biology, SWEDEN 151 85 Castro, Ana, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Chen, Shuibing, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Colombres, Marcela, CRCP Biomedical Center, Faculty of Biological Sciences, P. Catholic University of Chile, P.O. Box 114-D, Santiago, CHILE Ding, Sheng, Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA xxi

xxii

CONTRIBUTORS

Engel, Tobias, Centro de Biología Molecular “Severo Ochoa” CSIC/UAM. Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Facci, Laura, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, Essex, UNITED KINGDOM Farías, Ginny, CRCP Biomedical Center, Faculty of Biological Sciences, P. Catholic University of Chile, P.O. Box 114-D, Santiago, CHILE Fuertes, Ana, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Garrido, Juan José, Centro de Biología Molecular “Severo Ochoa”, CSICUAM, Cantoblanco, 28049 Madrid, SPAIN Gomez-Sintes, Raquel, Centro de Biología Molecular “Severo Ochoa” CSIC/UAM. Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Gould, Todd D., Laboratory of Molecular Pathophysiology, Bldg. 35, Room 1C-912, NIMH, NIH, Bethesda, MD 20892-3711, USA Grundke-Iqbal, Inge, Department of Neurochemistry, New York State Institute for Basic Research In Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314-6399, USA Hernandez, Félix, Centro de Biología Molecular “Severo Ochoa” CSIC/UAM. Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Inestrosa, Nibaldo C., CRCP Biomedical Center, Faculty of Biological Sciences, P. Catholic University of Chile, P.O. Box 114-D, Santiago, CHILE Iqbal, Khalid, Department of Neurochemistry, New York State Institute for Basic Research In Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314-6399, USA Jope, Richard S., Department of Psychiatry and Behavioral Neurobiology, Sparks Center 1057, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA Lindquist, Johanna M., AstraZeneca R&D, Södertälje, Disease Biology, SWEDEN 151 85 Lucas, José J., Centro de Biología Molecular “Severo Ochoa” CSIC/UAM. Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Manji, Husseini K., Laboratory of Molecular Pathophysiology, Bldg. 35, Room 1C-912, NIMH, NIH, Bethesda, MD 20892-3711, USA

CONTRIBUTORS

xxiii

Martín-Aparacio, Esther, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Martínez, Ana, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Medina, Miguel, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Monte-Millán, María, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Navarro, María L., NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Pérez-Puerto, Maria J., NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Simón, Diana, Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM, Cantoblanco, 28049 Madrid, SPAIN Skaper, Stephen D., Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, Essex, UNITED KINGDOM Ter Haar, Ernst, Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge MA 02139, USA Varea, Olga, Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM, Cantoblanco, 28049 Madrid Wandosell, Francisco, Centro de Biología Molecular “Severo Ochoa”, CSICUAM, Cantoblanco, 28049 Madrid, SPAIN Witherington, Jason, Department of Medicinal Chemistry, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research Limited, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UNITED KINGDOM Woodgett, James R., Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario M5G 1X5, CANADA

ABBREVIATIONS

Ab β-Amyloid peptide Abl-K Abelson tyrosine kinase ABP Actin-binding protein ACh Acetyl choline AD P-Tau AD cytosolic abnormally hyperphosphorylated Tau AD Alzheimer’s disease ADP Adenosine monophosphate AKAP A-Kinase anchoring protein ALS Amyotrophic lateral sclerosis AMPPNP Adenylylimidodiphosphate A-P Anterior-posterior APC Adenomatous polyposis coli ApoER Apolipoprotein E receptor APP Amyloid β precursor protein ATP Adenosine triphosphate AUC Area under the curve Axin Axis inhibitor BBB Blood-brain barrier BCL Breakpoint cluster BGTX Bungarotoxin BIO 6-bromoindirubin-3′-oxime xxv

xxvi

ABBREVIATIONS

BMP Bone morphogenetic protein BPNase Bisphosphate nucleotidase b-TRCP β-transducin-repeat-containing protein C/EBPa CCAAT/enhancer binding protein α CaMK II Ca2+ /calmodulin dependent kinase II cAMP Cyclic adenosine monophosphate CDK Cyclin-dependent kinase ChIP Chromatin immunoprecipitation Chk1 Checkpoint kinase 1 CHO Chinese hamster ovary cells CK Casein kinase CLb Clearance in blood CM Conditioned medium Cmax Maximum concentration CNS Central nervous system CoMFA Comparative molecular ﬁelds analysis COSY Correlation spectroscopy CRMP Collapsin response mediator protein CSNK1A Casein kinase 1 alpha C-Terminus Carboxy terminus CYP450 Cytochrome P450 DA Dopamine DAAM1 Disheveled activator of morphogenesis 1 DAG Diacylglycerol DALY Disability-adjusted life year DAT Dopamine transporter Dkk-1 Dickkopf-1 DMPK Drug metabolism and pharmacokinetics DN Dominant negative Dsh Disheveled DYRK Dual-speciﬁcity tyrosine-(Y)-phosphorylation regulated kinase E. coli Escherichia coli E10.5 Embryonic day 10.5 E18 Embryonic day 18 EBs Embryoid bodies ECS Electroconvulsive seizure ECT Electroconvulsive therapy EDG Endothelial diffreentiating gene

ABBREVIATIONS

xxvii

EGFP Enhanced green ﬂuorescent protein EGFR Epidermal growth factor receptor eIF Eukaryotic initiation factor ELISA Enzyme-linked immunosorbent assay EPSCs Epidermal stem cells ERa Estrogen receptor α ERK Extracellular signal-regulated protein kinase ERT Estrogen replacement therapy ESCs Embryonic stem cells FAD Familial Alzheimer’s disease FBPase Fructose 1,6-bisphosphastase FGF Fibroblast growth factor FP Fluorescence polarization Fpo% Percentage of oral bioavailability FRAT Frequently rearranged in advanced T-cell lymphomas FRET Forster resonance energy transfer FST Forced swim test FTD Frontotemporal dementia FTDP-17 Frontotemporal dementia with Parkisonism linked to chromosome 17 Fz Frizzled GFAP Gial ﬁbrillary acidic protein GLUT Glucose transporter GOLD Genetic optimisation of ligand design GS Glycogen synthase GSK Glycogen synthase kinase GTPases Guanosine tri-phosphate phosphatase HA Hemagglutin H-bond Hydrogen bond HD Hymenialdisine HER2 Epidermal growth factor receptor 2 HFs Hair follicles HIV-1 Human immunodeﬁciency virus type 1 HMBC Heteronuclear multiple bond correlation HMQC Heteronuclear multiple quantum correlation HNE 4-Hydroxy-2-nonenal HSCs Hematopoietic stem cells hSOD1 Human Cu/Zn-superoxide dismutase gene

xxviii

ABBREVIATIONS

HTS High-throughput screening IC50 Concentration that inhibits 50% ICM Inner cell mass ICV Intracerebroventricular IFE Interfollicular epidermis IGF-1 Insulin-like growth factor 1 IGF1R Insulin-like growth factor 1 receptor IMPase Inositol monophosphate phosphatase iNOS Inducible nitric oxide syntahse IP3 Inositol 1,4,5-trisphosphate IPPase Inositol polyphosphate 1-phosphatase IR Insulin receptor IRK Insulin receptor kinase IRS-1 Insulin receptor substrate 1 ISC Intestinal stem cells K15 Keratin 15 LEF Lymphoid enhancer-binding factor LIF Leukemia inhibitory factor LPA Lysophosphatidic acid LPS Lipopolysaccharide LRCs Label-retaining cells LRP Low-density lipoprotein receptor-related protein MAOIs Monoamine oxidase inhibitors MAP Microtubule associated protein MAPK Mitogen-activated protein kinase MARK Microtubule afﬁ nity regulating kinase MBD Microtubule binding domain mDab Mammalian disabled mECCs Murine embryonal carcinoma cells MEK MAPK-extracellular signal-regulated kinase MEK-1K MAP (mitogen-activated protein)/ERK (extracellular-signal regulated) kinase 1 MF Microﬁ lament Mg Magnesium MHB Midbrain-hindbrain boundary MIP Microtubule interacting protein Mkk Mitogen-activated protein kinase kinase MPCs Myogenic progenitor cells MSCs Mesenchymal stem cells

ABBREVIATIONS

MSV Murine sarcoma virus MT Microtubule NCSCs Neural crest stem cells NF Neuroﬁ lament NFkB Nuclear factor κB NF-L Neuroﬁ lament light chain NFT Neuroﬁbrillary tangles NGF Nerve growth factor NIDDM Non-insulin-dependent diabetes mellitus NMDA N-methyl-d-aspartic acid NMR Nuclear magnetic resonance NOD/SCID Non-obese diabetic/severe combined immunodeﬁcient NOESY Nuclear overhauser enhancement spectroscopy NPCs Neural precursor cells NSC Neural stem cell N-terminus Amino-terminus Nurr1 + Nuclear receptor-related factor 1 positive Ob Obese OGTT Oral glucose tolerance test OPG Osteoprotegerin P8 Postnatal day 8 PCR Polymerase chain reaction PDB Protein Data Bank PDGF Platelet-derived growth factor PDH Pyruvate dehydrogenase PDK Phosphoinositide-dependent protein kinase PDPKs Proline-directed protein kinases PEG Polyethylene glycol PGM Phosphoglucomutase PHF Paired helical ﬁ laments PI 3-kinase Phosphatidylinositol 3-kinase PI3K Phosphatidyl inositol 3 kinase PIP3 3,4,5 Phosphatidylinositol PK/PD Pharmacokinetics/pharmacodynamics PKA Cyclic AMP-dependent protein kinase PKA Protein kinase A PKB Protein kinase B (also termed Akt) PKC Protein kinase C

xxix

xxx

ABBREVIATIONS

PNS Peripheral neurous system PP Protein phosphatase PP1 Protein phosphatase 1 PP2A Protein phosphatase 2A PPAR g Peroxisome proliferator-activated receptor γ PrP Prion protein PS Presenilin PTB Phosphotyrosine-binding domain pTyr Phosphorylated tyrosine RANK Receptor activator of nuclear factor-KappaB RGC Retinal ganglion cell Rsk Ribosomal protein S6 kinase SAR Structure activity relationship SERM Selective estrogen receptor modulator SFRP1 Secreted Frizzled-related protein 1 sgg Shaggy SGs Sebaceous glands SH src Homology domain siRNA Small, interference ribonucleic acids SLF Steel factor SPA Scintillation proximity assay SR Serine/arginine rich sRNAi Short interfering RNA SSRIs Serotonin reuptake inhibitors t1/2 Half-life T2DM Type 2 diabetes mellitus T2K TNF Receptor associated factor 2 associated kinase TA Transit amplifying TCF T-Cell factor 1 TDZD Thiadiazolidinones TFE Triﬂuoroethanol TH + Tyrosine hydroxylase positive TNFa Tumor necrosis factor α TR-FRET Time-resolved forster resonance energy transfer TRK Tyrosine receptor kinase UDPG Uridine-5′-diphosphoglucose VEGF Vascular endothelial growth factor VLDLR Very low density lipoprotein receptor

ABBREVIATIONS

VM Ventral mesencephalon Vss Volume of distribution ZDF Zuker diabetic fatty zw Zeste-white

xxxi

PART I CELLULAR FUNCTIONS OF GSK-3

1 GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS James R. Woodgett Samuel Lunenfeld Research Institute, Toronto, Ontario

Contents 1.1 Introduction to GSK-3 1.2 A Brief History 1.2.1 Two Mammalian GSK-3 Molecules 1.2.2 Modes of Regulation 1.2.3 Structure and Function of GSK-3 1.2.4 GSK-3 Involvement in the Wnt Pathway 1.2.5 Signal Selectivity 1.2.6 GSK-3 Knockout Mice 1.2.7 GSK-3 and Glucose Metabolism 1.2.8 Neuronal Functions 1.3 Concluding Remarks

1.1

INTRODUCTION TO GSK-3

GSK-3 is a highly conserved protein kinase, and genes encoding the enzyme have been identiﬁed in every eukaryotic genome that has been investigated. In mammals, GSK-3 is encoded by two genes, termed GSK-3α and GSK-3β, that encode proteins of 51 and 47 kDa, respectively, and that share almost complete sequence identity between their protein kinase domains [1]. Indeed, the degree of conservation around the ATP binding site, the site at which Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

3

4

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

most small molecule inhibitors binds is essentially identical, making the likelihood of identiﬁcation of isoform-selective chemical inhibitors remote. That said, siRNAs have been designed that are selective [2]. As mentioned above, GSK-3 is unusual in that its protein kinase activity is high in resting cells. Cellular stimulation through a plethora of signaling pathways typically results in rapid and reversible reduction of its catalytic activity. Further many of the substrate proteins of GSK-3 are functionally inhibited when phosphorylated by this kinase. As a consequence signals that suppress GSK-3 activity typically act to induce the function of GSK-3 substrates. Genetic analyses and the use of selective inhibitors have shown that GSK-3 plays critical roles in development, metabolic homeostasis, neuronal growth and differentiation, cell polarity, and cell fate, as well as in modulation of apoptotic potential. Before decisions are taken with respect to the use of agents that target speciﬁc functions of GSK-3, it is necessary to understand how GSK-3 is regulated in its many roles in the cell. While inhibition of GSK-3 may be desired in one context, say, in preventing neuronal cell death in a degenerative disorder, it could have adverse side effects, such as promotion of tumorigenesis, if the same inhibition of activity affects another function of GSK-3—such as the regulation of cell division. GSK-3 is ubiquitously expressed and constitutively active in unstimulated tissues. Regulation of GSK-3 substrate phosphorylation tends to occur either by suppression of GSK-3 or by reducing substrate access. Each of these modes has a place in tailoring the cellular response to the signal to which the kinase is responding. This introductory chapter serves as a primer for the means by which cells control GSK-3 regulation, the signaling pathways in which it participates, and the consequences of its dysregulation.

1.2 A BRIEF HISTORY GSK-3 was puriﬁed as one of ﬁve protein kinase activities found to phosphorylate glycogen synthase (GS) in fractionated extracts of rabbit skeletal muscle [3,4]. GSK-3 phosphorylates four serine residues on GS, resulting in its inactivation. The phosphorylated serine residues (at positions 641, 645, 649, and 653) are spaced four amino acids apart and are preceeded by a further serine at position 657. This latter residue is not targeted by GSK-3, but by casein kinase-II, and when S657 is phosphorylated, it forms a priming site for GSK-3: Ala640 -Ser(P)-Val-Pro-Pro-Ser(P)-Pro-Ser-Leu-Ser(P)-Arg- His-Ser-Ser(P)-Pro-His-Gln Ser657

Indeed, GSK-3 is incapable of phosphorylating GS until it has been phosphorylated at S657. This phenomenon of primed phosphorylation is a common motif among GSK-3 targets (see below). Despite its initial discovery as a

5

A BRIEF HISTORY

metabolic enzyme regulator, GSK-3 is by no means restricted to a role in glycogen metabolism. Indeed, the protein targets a wide variety of proteins involved in signaling, metabolism, structural proteins and a remarkable number of transcription factors (see Table 1.1). Despite its name, it is worth repeating that GSK-3 plays a far more pleiotropic role than ﬁ rst imagined, although its regulation of glycogen metabolism may well have important therapeutic value in type 2 diabetes (see below).

TABLE 1.1 Substrates of GSK-3 Substrate

Effect of GSK-3 Phosphorylation Transcription Factors

BCL-3 (breakpoint cluster 3) β-catenin cAMP response element binding protein CCAAT/enhancer-binding protein α/β c-Jun c-Myc GATA 4 Glucocorticoid receptor Heat shock factor-1 Hypoxia inducible factor 1 Microphthalmia-associated transcription factor Neuro-D (Xenopus) NF-κB (p65 and p105)

N-myc downstream regulated gene 1 Notch Nuclear factor of activated T-cell c p53 Timeless

Targets for proteasomal degradation Targets for proteasomal degradation Increased transcription factor activity Enhanced activity of CEBPα; reduced activity of CEBPβ Decreased DNA binding and transactivation Targets for degradation Induces nuclear export Inhibits transcription factor activity Inactivates transcription factor activity Downregulates by reducing accumulation Increased binding to tyrosinase promoter Inhibits transcription factor activity Negatively regulates basal p65 activity; stabilizes p105 under resting conditions but destabilizes after TNFα treatment Unknown Downregulates activity; destabilizes Decreased DNA binding; increased nuclear export Activates transcriptional activity Increased heterodimerization with clock gene called period or increased nuclear transport

Structural Proteins CRMP Dynamin-like protein

Reduced microtubule polymerization and axonal growth Unknown

6

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

TABLE 1.1

Continued

Substrate

Effect of GSK-3 Phosphorylation

High molecular weight mucin-like glycoprotein (DF3/Muc) Kinesin light chain Microtubule associated protein 1B Microtubule associated protein 2 Neural cell-adhesion molecule Ninein Tau Telokin / kinase-related protein Adenomatous polyposis coli

Decreases binding to β-catenin (junctional) Inhibits anterograde vesicular movement Destabilizes microtubules Prevents microtubule bundling Unknown Unknown Reduced microtubule binding; decreased microtubule stability Unknown Increased β-catenin binding; decreased microtubule binding

Metabolism and Signaling Amyloid precursor protein ATP-citrate lyase Axin Cubitus interruptus cAMP-dependent protein kinase Cyclin E eIF-2B translation factor Glycogen synthase Heterogeneous nuclear ribonucleoprotein D Inhibitor-2 Insulin receptor substrate 1 Nucleoporin p62 p21 Presenilin 1 (PS1) Protein phosphatase 1 Protein phosphatase inhibitor-2 Pyruvate dehydrogenase

1.2.1

Increases cleavage by γ-secretase and production of Aβ peptide Inactivates enzyme Increased afﬁ nity for β-catenin and increased Axin stability Causes proteolytic cleavage Affects afﬁ nity of regulatory subunits Targets for degradation Inhibits activity Inhibits activity Inhibits transactivation activity Activates phosphatase Inhibits insulin receptor signaling Modulates nuclear pore Increases proteasomal degradation Increases degradation of C-terminal PS1 fragment Activates phosphatase Activates phosphatase Inactivates enzyme

Two Mammalian GSK-3 Molecules

There are two genes that encode GSK-3 that are localized on distinct chromosomes in mouse and human genomes [1,5]. These two genes, designated GSK-3α and GSK-3β, are almost identical within their protein kinase domains (98%) but differ within their N- and C-terminal regions. The largest structural difference between the two is an amino-terminal, glycine-rich region

A BRIEF HISTORY

7

speciﬁc to GSK-3α and this accounts for the 4 kDa difference in the protein masses. A splice isoform of GSK-3β is expressed speciﬁcally in neuronal tissues and consists of a 13 amino acid loop inserted into the large lobe of the kinase domain, within the substrate-binding region [6]. Whether this isoform has a distinct function has yet to be determined. In some species such as yeast and plants there are multiple GSK-3 genes, whereas others only express a single orthologue. For example, in Drosophila, a single GSK-3 gene, termed shaggy/zeste-white3 (sgg/zw3), undergoes differential splicing to form several distinct polypeptides [7]. Isolation of alleles that selectively disrupt particular splice variants has revealed distinct biological functions during Drosophila development (A. Manoukian, in preparation). In mammals, GSK-3α and β are each expressed widely at both the RNA and protein levels although some tissues show preferential levels of the two proteins. Neither gene appears to be acutely regulated at the transcriptional level and both proteins have relatively long half-lives. In other words, the proteins are controlled posttranslationally, largely through protein:protein interactions or by posttranslational regulation. 1.2.2

Modes of Regulation

Both mammalian GSK-3 proteins are regulated by phosphorylation within their amino-terminal domain: GSK-3α is phosphorylated at S21, while GSK3β is phosphorylated at S9. GSK-3 activity toward exogenous substrates is inhibited by phosphorylation at these residues. Both GSK-3 proteins are also tyrosine phosphorylated within their T-loop region at Y279/216 (GSK-3α and β, respectively) [8]. This is located in an analogous position to the activating phosphorylation of tyrosine (and threonine) in the mitogen-activated protein kinase (MAPK) superfamily. However, in GSK-3, modiﬁcation of this residue appears to be the result of an autophosphorylation reaction, likely soon after initial synthesis rather than by a distinct upstream kinase (e.g., Mek or Mkks) [9] (see below). Furthermore the degree of phosphorylation of this tyrosine is high in most cells. Several protein kinases are capable of phosphorylating and inactivating GSK-3 at the N-terminal domain site. The most studied is protein kinase B (PKB/Akt) because this enzyme represents the predominant activity that performs this modiﬁcation in response to insulin [10]. Insulin binding to its receptor induces the tyrosine phosphorylation of speciﬁc IRS adaptor proteins which then recruit various SH2 and PTB domain proteins to the plasma membrane, including several isoforms of phosphatidylinositol 3′ kinase (PI3K). The PI3K then phosphorylates the 3′ position of the inositol ring of phosphatidylinositol 4,5-phosphate, leading to the formation of 3,4,5 phosphatidylinositol (PIP 3). This speciﬁc phospholipid is limited to the membrane and has a high afﬁ nity for proteins harboring pleckstrin homology domains. Two such molecules are phosphoinositide-dependent protein kinase-1 (PDK1) and PKB. Upon PIP 3 -dependent co-localization of both PDK1 and PKB at

8

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

the membrane, PDK1 phosphorylates and activates PKB (at serine 308 in PKBα) leading to its activation. A prominent target of active PKB is GSK-3, which is inhibited once phosphorylated [11,12]. Other protein kinases that target the N-terminal domain of GSK-3 include cyclic AMP-dependent protein kinase (PKA) [13,14], atypical protein kinase C’s (PKC) [15] and p90Rsk [16]. Control of GSK-3 activity thus occurs by complex mechanisms that are each dependent on speciﬁc signaling pathways. Furthermore GSK-3 appears to be a cellular nexus, integrating several signaling systems, including several second messengers and a wide selection of cellular stimulants. 1.2.3

Structure and Function of GSK-3

As introduced above, GSK-3 exhibits a strong preference for phosphorylating protein substrates that have been previously phosphorylated or “primed” [17]. The priming phosphorylation sites lie carboxy-terminal to the phosphorylation sites that are targeted by GSK-3. In several substrates the phosphate added by GSK-3 upstream of the phosphorylated priming site often serves as the priming site for additional phosphorylation of a serine/threonine four residues upstream, leading to serial phosphorylation. The requirement for priming phosphorylation raises the possibility that regulation of the priming kinase may “gatekeep” the GSK-3 phosphorylation of a protein. This would explain how GSK-3 phosphorylation of proteins could be regulated if GSK-3 activity is constitutive. However, this does not appear to be an active mode of control since most priming phosphorylation sites are constitutively phosphorylated and their stoichiometry of phosphorylation does not appear to vary upon cellular stimulation. Three groups solved the crystal structure of GSK-3β and this revealed insight into the role of priming and regulation of the protein kinase [18–20]. Like typical protein kinases, GSK-3 comprises an amino-terminal lobe composed mostly of β-sheets and a carboxy-terminal lobe that is predominantly α-helical. As mentioned above, the activation or T-loop of GSK-3 is only phosphorylated at a single tyrosine. Although tyrosine phosphorylation might be envisioned as being sufﬁcient to open the substrate binding site, there appear to be no structural constraints to prevent an open conformation in the unphosphorylated state [19]. Indeed, removal of the phosphorylated tyrosine does not completely inactivate the enzyme, causing only a reduction in Vmax. The role of the “missing” T-loop phospho-threonine that is typically present and required to be phosphorylated in the MAP kinase superfamily appears to be substituted for by the phosphorylated residue of a primed substrate that binds to a positively charged pocket comprised of R96, R180, and K205 (residues for GSK-3β). This docking of the primed phosphate group not only optimizes the orientation of the two kinase lobes but also positions the substrate within the catalytic cleft to permit efﬁcient phosphorylation at a serine or threonine residue 4 amino acids upstream of the priming site to occur. Although some substrates do not require priming phosphorylation, these

A BRIEF HISTORY

9

rarer substrates often display negatively charged residues at or near the primed phosphate position and thus may mimic a phosphorylated residue. The crystal structure of GSK-3 also helped in explaining how amino terminal domain serine phosphorylation is inhibitory, when it occurs so distal to the catalytic site. Phosphorylation of S9 of GSK-3β or S21 of GSK-3α creates a primed pseudosubstrate that can enter the positively charged phosphate pocket via intramolecular association. By doing so, the N-terminal domain prevents phosphorylation of exogenous substrates since the catalytic groove will be occupied with the N-terminal domain peptide that is slow to release, since it does not contain a phosphorylatable residue in the catalytic site of GSK-3. Although an intramolecular interaction, the afﬁ nity of the phosphorylated N-terminal domain is low enough that phosphorylated primed substrates, at high enough concentration, can compete off the inhibitory polypeptide [21]. However, in vivo the inhibition is most likely only reversed by dephosphorylation of the N-terminal residue, possibly by a phosphatase termed Laforin [22]. A short, 8 residue peptide matching the phosphorylated N-terminus of GSK-3β (NTptide-8) selectively inhibited GSK-3 phosphorylation of primed substrates [21], raising the possibility that small molecules that are targeted to the positively charged pocket of GSK-3’s kinase domain may be useful in the selective inhibition of phosphorylation of GSK-3 substrates. 1.2.4 GSK-3 Involvement in the Wnt Pathway Although the N-terminal phosphorylation is the primary means by which growth factors and insulin inhibit GSK-3, this mode of control plays no role in regulating GSK-3 function by the Wnt pathway (see below; [10,23,24]). Wnts are a family of secreted, glycosylated ligands that bind members of the Frizzled family of serpentine receptors. Fruit ﬂ ies have a single Wnt-related gene termed wingless that mediates cell fate speciﬁcation and patterning during development. The situation is far more complex in mammals where 19 genes encoding Wnts proteins play key roles in cell growth, differentiation, migration, and cell fate determination. The prototypical Wnt, Wnt1, was initially isolated as a site of common integration for mouse mammary tumor virus. Selective expression of Wnt1 in mouse mammary epithelial cells causes mammary tumors [26]. Abrogated expression of Wnts has also been implicated in various human tumors. Speciﬁc Wnt proteins elicit distinct downstream effects within cells. Some appear inhibitory, while others affect planar polarity and actin remodeling. A role for GSK-3 in Wnt signaling appears restricted to the “canonical” Wnt signaling pathway, which is most closely associated with human cancers and which relies on tight control of the cytoplasmic levels of β-catenin as a transcriptional regulator. In unstimulated cells, GSK-3 negatively regulates signaling through the Wnt pathway by directly phosphorylating β-catenin at three residues toward the amino terminal end of the protein, S33, S37, and T41. Phosphorylation of

10

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

S33 and S37 target β-catenin for ubiquitinylation by the E3 ubiquitin ligase βTrCP [26,27], and subsequent degradation via the 26S proteasome. Under these conditions the cytoplasmic and nuclear concentrations of β-catenin are maintained at extremely low levels. In response to exposure to speciﬁc Wnt ligands, the phosphorylation of β-catenin is inhibited, allowing the now stabilized β-catenin to accumulate in the cytosol and nucleus and to interact with members of the LEF1/TCF family of DNA binding proteins. This results in transactivation of genes that harbor TCF binding sites. GSK-3 mediated phosphorylation of β-catenin requires primed phosphorylation, provided by casein kinase-1 phosphorylation of S45. However, the level of phosphorylation of S45 does not appear to change upon Wnt stimulation, whereas the three sites targeted by GSK-3 are signiﬁcantly reduced. GSK-3 only interacts with β-catenin within the context of a large protein complex that comprises GSK-3, β-catenin, a scaffolding protein called Axin, and the tumor suppressor, adenomatous polyposis coli (APC). Indeed, GSK-3 only phosphorylates β-catenin when both molecules are simultaneously bound to Axin [28,29]. APC, β-catenin, and Axin are frequently mutated in human tumors, and hence activation of this pathway plays a signiﬁcant role in cancer. For example, loss-of-function mutations in APC occur in nearly 70% of sporadic colon cancers and gain-of-function mutations in β-catenin are associated with a further 15%. Mutations in β-catenin tend to be localized to the region harboring the GSK-3 and casein kinase 1 (CK-I) phosphorylation sites and reduce or block phosphorylation of the sites that target the molecule for ubiquitinylation and degradation leading to chronic stabilization of the protein. Mutations in Axin are found in hepatocellular carcinomas amongst others (reviewed in Salahshor [30]). Axin comprises a string of protein– protein interaction domains that independently bind each of the components of the multi-protein complex, including GSK-3 (both α and β), APC, protein phosphatase 2A, and Disheveled. Axin also contains a dimerization domain. In addition to phosphorylating β-catenin, GSK-3 also phosphorylates Axin and APC at several sites. GSK-3-mediated phosphorylation of Axin promotes its stability but has no effect on the binding of β-catenin to Axin [31]. APC phosphorylation increases its afﬁ nity for binding to β-catenin that helps balance the association between APC and β-catenin over the wide concentration range of β-catenin that occurs in the absence or presence of a Wnt signal [32]. GSK-3 phosphorylation of Axin occurs within the β-catenin binding domain of Axin and alteration of these residues to alanine impairs β-catenin binding to Axin in vitro and reduces Wnt regulation of β-catenin. Wnt stimulation leads to a reduction in Axin phosphorylation, and this may contribute to Wnt-mediated stabilization of β-catenin [33], a functional consequence similar to direct inhibition of GSK-3 activity [34–36]. In mouse L929 cells the binding of GSK-3 to Axin was reduced within minutes of exposure to Wnt3a, and this effect was dependent on G (α0 or αq) protein function as it was inhibitable by pertussis toxin [37]. Wnt-induced dissociation of GSK-3 from

A BRIEF HISTORY

11

the Axin/APC complex would provide a means by which β-catenin phosphorylation could be reduced in response to the ligand but does not address the mechanism by which this occurs (see below). As mentioned above, GSK-3 is not the only protein kinase capable of phosphorylating β-catenin. Indeed, GSK-3 requires β-catenin to be previously phosphorylated by CK-Iα at S45 before it becomes a GSK-3 substrate. This raises the possibility that Wnt-induced inhibition of CK-I activity could lead to dephosphorylation of priming phosphate at S45, thus preventing GSK3 phosphorylation of the residues on β-catenin that signal ubiquitinylation (S33 and S37). However, genetic experiments in Drosophila indicate that CK1-mediated phosphorylation of the orthologous residue (S56) of the ﬂy orthologue of β-catenin, Armadillo, is insensitive to Wingless treatment [38]. Furthermore, in mouse embryonic stem cells that have been engineered to delete both isoforms of GSK-3, phosphorylation of β-catenin at S45 appears constitutive and insensitive to Wnt3a (B. Doble, S. Patel and J. Woodgett, in preparation). A distinct CK-I isoform has recently been implicated in positive regulation of Wnt signaling, again in concert with GSK-3 [39–41]. CK-Iγ is an unusual isoform of this family of kinases in that it contains a C-terminal palmitoylation motif that effectively anchors it to the plasma membrane. The Frizzled family of serpentine Wnt receptors require single membrane-pass co-receptors termed LDL-receptor-related proteins 5 and 6 (LRP5 and 6) to transduce a Wnt signal [42]. LRP5 and 6 each become phosphorylated on ﬁve repeats of a PPPSP motif within their C-terminal domains in response to Wnt binding and mutation of these sites inhibits downstream signaling. The PPPSP motifs are each preceded by a further phosphorylatable residue (either T or S) generating an extended motif: PPPSPxS/T. The C-terminal residue of this sequence is targeted by casein kinase-1γ, whereas the PPPSP motif is targeted by GSK-3. While GSK-3 clearly plays a negative role in the Wnt pathway by promoting proteolysis, it also plays a positive role at the level of LRP5/6, since ﬁbroblasts engineered to lack both alleles of GSK-3 are defective in LRP6 (and presumably LRP5) phosphorylation [39]. Dual phosphorylation of the PPPSPxS/T motif in LRP5/6 creates a high-afﬁ nity binding site for Axin. Assembly of the above data allows for several plausible models by which Wnt may lead to the stabilization of β-catenin. The initial events are in common. Wnt binding to Frizzled and LRP5 or 6 initiates a conformational change in the receptor complex. This facilitates rapid phosphorylation of the PPPSPxS/T motifs by GSK-3 and casein kinase 1γ that then leads to recruitment of Axin to the receptor complex. This event may cause GSK-3 associated with Axin to be displaced, effectively precluding further phosphorylation of β-catenin; the β-catenin then accumulates, triggering transcriptional changes in the nucleus. The timescale of the response is worth noting. The Wntinduced disruption of the Axin/GSK-3 complex occurs within minutes. However, β-catenin takes several hours to accumulate, since its transcription

12

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

is not induced. Moreover one of the primary transcriptional targets of nuclear β-catenin (when associated with the LEF/TCF DNA binding proteins) is Axin-2, a homologue of Axin [30]. Induction of this gene product acts to antagonize Wnt signaling by increasing the capacity of cells to degrade βcatenin by recruiting more GSK-3. The system is therefore hardwired to shut down after initial activation. Only when the pathway is genetically abrogated is chronic signaling possible, with disastrous consequences. Whether Wnt ligation alters the activity of either or both of the two kinases acting on the LRP motif is unclear. It is possible that Wnt binding causes unfolding of the PPPSPxS/T motifs such that they are now recognized by the two kinases. It is also possible that the fraction of GSK-3 associated with Axin (which is <20% of the total GSK-3 within a cell) is decoyed by the newly unveiled LRP motifs such that phosphorylation of these sites is mutually exclusive with phosphorylation of β-catenin (and Axin and APC). There remain several important questions. Why are there multiple PPPSPxS/T motifs in the LRPs? Mutational analysis suggests only one is required for signaling [42]. Does the multiplicity cause Axin clustering or do other proteins involved in Wnt signaling bind to the phosphorylated motif? What is the role of Disheveled? Genetic epistasis analysis in Drosophila has clearly demonstrated a need for this protein in both canonical and noncanonical Wnt signaling. A more teleological question is why nature developed such as complex and unusual mechanism of interdependencies for Wnt activation? All of the intracellular components of the pathway have distinct functions, independent of the Wnt pathway. The answer may be trivial in that it may represent the ultimate example of reduce, reuse, recycle, but parsimony is not a common trait in biological processes as important as signal transduction. 1.2.5

Signal Selectivity

As discussed earlier, the primary means by which mitogens and hormones such as insulin negatively regulate GSK-3 is via N-terminal phosphorylation at S21 or S9. However, this mechanism does not appear to play a role in the regulation of GSK-3 in the Wnt pathway. For example, mutants of Drosophila GSK-3 that cannot be phosphorylated at the N-terminal site do not have Wingless pathway phenotypes [43]. Furthermore knock-in mice in which both S9 of GSK-3β and S21 of GSK-3α had been converted to alanine showed no developmental defects, suggesting that Wnt functions were normal [44]. An emerging theme in signaling is the sequestration of distinct pools of signaling molecules within organizing complexes such as A-kinase anchoring proteins (AKAPs) and other scaffolding proteins. Indeed, cells partition subpopulations of GSK-3 such that growth factors and hormones that inhibit GSK-3 by inducing N-terminal phosphorylation, have no effect on β-catenin stabilization. By contrast, exposure of cells to Wnts does not cause changes in GSK-3 substrates that are regulated by growth factors and hormones such as intermediary metabolism. Clearly, this is a critical property of GSK-3 regulation

A BRIEF HISTORY

13

given the consequences of ectopic or inappropriate stabilization of β-catenin on tumorigenesis. 1.2.6

GSK-3 Knockout Mice

Mice that are deleted for both alleles of GSK-3β die late during embryogenesis. The most pronounced defect is proliﬁc apoptosis of hepatocytes due to enhanced sensitivity to TNFα [45]. Other effects are revealed upon rescue of this primary defect by administration of anti-TNFα antibodies or breeding to mice lacking TNF receptor 1 [45 and L. Kockeritz, unpubl.]. This acute hepatocyte sensitization is reminiscent to knock out of components of the NF-κB pathway such as RelA and T2K [46,47]. Furthermore pre-treatment of hepatocytes with LiCl, which inhibits GSK-3, synergizes TNFα-induced killing [48]. TNFα treatment of wild-type ﬁbroblasts induces NF-κB activation but not in cells that lack for GSK-3β [45]. In one study GSK-3β was shown to phosphorylate p65 in vitro [48]. p65 has also been reported to be phosphorylated at S468 by GSK-3β in vivo, but this paralleled a decrease in NF-κB activity [49]. Inhibition of GSK-3 in pancreatic tumor cells signiﬁcantly reduced growth and survival and inhibited NF-κB activation, and these authors also reported elevated GSK-3 activity at the leading edge of primary pancreatic tumors [50]. Mice lacking both alleles of GSK-3α have recently been generated in our laboratory as well as mice containing conditional alleles of both GSK-3α and GSK-3β. Parallel studies will allow evaluation of the roles of these protein kinases in speciﬁc tissues through crossing with tissue-speciﬁc Cre recombinase deletor strains. 1.2.7

GSK-3 and Glucose Metabolism

Insulin is a critical regulator of glucose homeostasis. In peripheral tissues insulin promotes the storage of glucose by promoting the rate of glucose entry into tissues and through the activity of glycogen synthase (GS), the rate-limiting enzyme of glycogen synthesis. Uptake of glucose is mediated by insulininduced fusion of glucose transporter proteins (e.g., GLUT 4) from intracellular compartments to the plasma membrane [51,52]. GS activity is regulated by phosphorylation at speciﬁc sites. Insulin stimulates glycogen synthesis by inducing the dephosphorylation of GS primarily at four residues targeted by GSK-3 [53]. Inhibition of GSK-3 is through the PI3’K-dependent phosphorylation of the N-terminal domain serine residues (S21 in GSK-3α and S9 in GSK-3β). Expression of wild-type or S9A mutant GSK-3β reduces basal as well as insulin-stimulated glycogen synthase activity in L6 muscle and 3T3-L1 adipocyte cell lines [54,55]. Mice have recently been generated in which the inhibitory phosphorylation sites were converted to alanine and then “knocked in” to replace the endogenous gene sequences by homologous recombination [44]. This experiment demonstrated that inactivation of GSK-3 is the major

14

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS

mechanism by which insulin stimulates the activity of GS in skeletal muscle. Various small molecule inhibitors of GSK-3 have been demonstrated to have insulin-mimetic properties. Lithium chloride promotes glucose transport and glycogen synthesis in adipocytes and muscle cells [56–62]. Two small molecule ATP-like inhibitors, SB 216763 and SB 415286, both increase glycogen deposition in human liver cells and mimic the property of insulin to control hepatic gene transcription [63,64]. In patients with type 2 diabetes mellitus (T2DM), circulating insulin levels are unable to maintain normoglycaemia due to desensitization of insulin responses in target tissues. The typical insulin resistance associated with T2DM is characterized by signal uncoupling of the insulin receptor from key downstream components as well as by the failure of pancreatic β-cells in compensating for the increased insulin resistance through increased secretion of insulin. These two defects lead to loss of control of glucose homeostasis, resulting in bouts of hyperglycaemia and, when uncontrolled for prolonged time, to complications including kidney nephropathy, blindness, and metabolic syndrome. There are no reports of genetic defects in either of the two isoforms of GSK-3 that have been associated with T2DM [5], likely because the two genes are largely redundant—requiring multiple mutations. However, GSK-3 activity, as well the levels of expression, are elevated in insulin resistant rodents as well as in skeletal muscle of type 2 diabetes patients [65,66]. GSK-3 acts as a negative regulator of insulin signaling via phosphorylation of insulin receptor substrate 1 (IRS-1) at S332 that inhibits IRS-1 function, either by suppression of IRS-1 protein levels or by making it a poorer substrate for tyrosine phosphorylation by the insulin receptor [67,68]. Consistent with these data, overexpression of a constitutively active form of GSK-3β (S9A) in skeletal muscle of mice reduces glucose tolerance, elevates plasma insulin levels, and reduces IRS-1 protein expression and muscle glycogen content [69]. Pharmacological inhibition of GSK-3 activity is thus expected to act as an insulin sensitizer for patients with T2DM, and there are several active drug development programs working to this end. 1.2.8

Neuronal Functions

An important substrate of GSK-3 in neurons is a protein termed collapsin response mediator protein 2 (CRMP2) [70–72]. CRMP binds to microtubules its phosphorylation by GSK-3 inhibits this binding, which promotes increased microtubule polymerization and axonal outgrowth. Expression of a mutant form of CRMP that is resistant to GSK-3 phosphorylation causes the formation of multiple axons. Adenomatous polyposis coli is also a microtubulebinding protein and is found in neurons at the plus ends of microtubules. As with CRMP2, phosphorylation of APC by GSK-3 acts to destabilize the microtubule threads [73]. Several other brain-enriched structural proteins are GSK-3 substrates (see Table 1.1), including microtubule-associated protein 1b

CONCLUDING REMARKS

15

(MAP1b) and Tau. Elevated phosphorylation of Tau is associated with formation of neuroﬁbrillary tangle precipitates—a pathology observed in Alzheimer’s disease [74]. GSK-3 therefore appears to play a complex role in coordination of microtubules, especially in the brain (although similar roles in nonneuronal cells is also likely). A further connection with Alzheimer’s disease was determined by the ﬁ nding that inhibition of GSK-3 reduced the production of the Aβ peptide from amyloid precursor protein [75]. Aberrant Aβ peptide leads to the formation of telltale amyloid plaque deposits in the brains of patients with Alzheimer’s disease through the action of a protease termed γ-secretase. These data suggest that GSK-3 modulates this enzyme activity, inhibitors of which are also in development for this neurodegenerative disease. These neuronal targets likely each play a role in the phenotypes associated with dysregulation of GSK-3 in various pathophysiological conditions. Several conditions that promote neuronal cell death result in GSK-3 activation. These include the amyloid precursor protein fragment [74], polyglutamine [76], Aβ [77], and prion peptides [78]. Dysregulated GSK-3 likely exerts its effects by interfering with growth cone and axonal morphologies through disruption of microtubules, and it is noteworthy that many brain-enriched structural proteins are substrates of GSK-3 (see above and Table 1.1).

1.3

CONCLUDING REMARKS

GSK-3α and β have a plethora of functions that complicate assessment of these molecules as a target for therapy. There are multiple important pathological conditions in which GSK-3 is dysregulated including neurological disorders such as bipolar disorder, sleep disorders, Alzheimer’s disease, and T2DM. In each of these conditions, inhibition of GSK-3 should provide relief of the driving forces of the diseases. The major concern, however, is that chronic inhibition of GSK-3 can also lead to activation of targets that are proliferative such as certain transcription factors and β-catenin, which is a human oncogene. To counter this concern, there are indications that the levels of inhibition required for effects on metabolic or neurological behaviors are much lower than required for stabilization and activation of β-catenin. Only 25% inhibition of total GSK-3 (as achieved in mice heterozygous for GSK-3β) is sufﬁcient to phenocopy behavioral effects of lithium treatment in mice and to have effects on amphetamine responses. [79]. Therapeutically effective doses of lithium (ca. 1 mM) for bipolar disorder result in 20% to 25% inhibition of GSK-3, and such patients show no documented evidence of enhanced colon or other cancers. Indeed, mice that have enhanced Wnt signaling and multiple intestinal polyps due to mutation of one allele of APC (“min” mice) do not exhibit increases in polyp formation when treated with lithium [80]. The GSK-3β heterozygote mice do show enhanced Wnt signaling in speciﬁc brain regions likely due to an increased sensitivity to endogenous Wnt signals

16

GLYCOGEN SYNTHASE KINASE 3: AN INTRODUCTORY SYNOPSIS Insulin

Frizzled

Padins 4,5 P2

Padins 3,4,5 P3 p85

pY

pY

pY

p110

LRP5/6

LRP5/6

PDK1 P

P1-3 Kinase

pY

nin

β-caPte P P

S

PKB

P

PY

SP PY

GS

atenin P

APC

β-cate

nin

β-cate

Active Destruction Complex nin

β-cate

P P

Proteasome

Glycogen(n + 1)

P P

Axin

nin

cate β-cate ββ β-cat

GSK-3 APC

GSK-3 (inactive)

PY

nin

β-cate

nin

β-cate

CKI

Glycogen(n) + UDP-Glucose

S

Axin

pY

IRS1/2

Frizzled

Wnt

Disrupted Destruction Complex

Nucleus

MAP

S

P P

GSK-3

GSK-3

TF

S

S

PY

nin

β-cate

PY

TCF P

P

PY

Insulin Receptor

GSK-3

GS

P

TF

Figure 1.1 Various pathways and modes of GSK-3 regulation and function. Inactivation of GSK-3 can occur through serine 9/21 phosphorylation as depicted in the upper left-hand corner, where the kinase PKB, a component of the PI3’K pathway is shown phosphorylating and inactivating GSK-3. Glycogen synthase (GS), as is typical of GSK-3 substrates, is inactive when phosphorylated by GSK-3. PKB-mediated inactivation of GSK-3 allows dephosphorylation and activation of GS that can then synthesize glycogen. In the Wnt pathway, GSK-3, in combination with the scaffolding protein Axin, the priming kinase CKI, and adenomatous polyposis coli (APC), work in concert to phosphorylate β-catenin, targeting it for proteasomal degradation. Wnt treatment causes β-catenin to escape phosphorylation by GSK-3 through an, as yet, unclear mechanism involving Wnt-dependent phosphorylation of LRP5/6, a coreceptor for Wnt, by GSK-3, and by consequent disruption of the complex required for β-catenin destruction. Note that regulation of GSK-3 in the Wnt pathway is independent of serine 9/21 phosphorylation. The β-catenin that escapes GSK-3 phosphorylation accumulates in the cytosol and translocates to the nucleus where it can activate TCF-mediated gene transcription. GSK-3 phosphorylates and inactivates several transcription factors (TF), targeting some for proteasomal degradation and others for nuclear export. GSK-3 also phosphorylates several microtubule-associated proteins (MAPs) such as Tau and MAP1B. See color plates.

[79]. However, as discussed above, the Wnt pathway triggers its own inhibition through the transcriptional induction of Axin 2 and this feedback loop is intact in cells with partial reduction of GSK-3. Insulin treatment causes about 50% inhibition of GSK-3 activity—a level sufﬁcient to activate glycogen synthase in skeletal muscle. Various types of

REFERENCES

17

ATP-competitive inhibitors of GSK-3 that have been developed cause insulin sensitization effects in cells as well as in rodent models of diabetes. Due to the reversible action (noncovalent) of the inhibitors, it is difﬁcult to measure the inhibition percentage of GSK-3 in vivo. However, use of phospho-speciﬁc antibodies to GSK-3 substrates such as glycogen synthase and β-catenin has allowed estimation of the degree of inhibition of this kinase in cells and tissues. In the case of β-catenin, GSK-3 inhibition promotes dephosphorylation and subsequent accumulation of the transcription factor. Using one class of inhibitors, no elevation in β-catenin levels was observed in diabetic treated with the inhibitor. However, the GSK-3 blocker activated glycogen synthase and hepatic glycogen synthesis [81,82]. These data suggest that mild inhibition of GSK-3 (ca. 30–40%) may be sufﬁcient for insulin-resensitization of muscle and liver tissues in T2DM, and that a much stronger degree of inactivation (>75%) of GSK-3 may be required to invoke growth-promoting effects. These effects are likely to be transient as well. Given the chronic nature of the diseases that might beneﬁt from GSK-3 blockade, clinical interventions employing GSK-3 inhibitors will require longterm monitoring for evidence for oncological effects. Nonetheless, there is clearly signiﬁcant promise for anti-GSK-3 drugs for alleviating several highly intractable diseases. The more that is understood about this unusual but remarkable protein kinase, the better equipped we will be in determining the safe use and limits of its inhibitors. ACKNOWLEDGMENTS JRW is a CIHR Senior Investigator and an International Scholar of the Howard Hughes Medical Institute and receives research grants from the Canadian Institutes of Health Research and from the Banting and Best Diabetes Centre (Toronto). Special thanks to the GSK-3 team in the laboratory: Brad Doble (who also designed Fig. 1.1), Satish Patel, Lisa Kockeritz, Ioana Miron, and Katrina MacAulay. REFERENCES 1. Woodgett J.R. (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J., 9, 2431–2438. 2. Yu J.Y., Taylor J., DeRuiter S.L., Vojtek A.B., Turner D.L. (2003). Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol. Ther., 7, 228–236. 3. Embi N., Rylatt D.B., Cohen P. (1980). Glycogen synthase kinase-3 from rabbit skeletal muscle: Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem., 107, 519–527. 4. Hemmings B.A., Yellowlees D., Kernohan J.C., Cohen P. (1981). Puriﬁcation of glycogen synthase kinase 3 from rabbit skeletal muscle: Copuriﬁcation with the

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63. Coghlan M.P., Culbert A.A., Cross D.A., Corcoran S.L., Yates J.W., Pearce N.J., Rausch O.L., Murphy G.J., Carter P.S., Roxbee Cox L., Mills D., Brown M.J., Haigh D., Ward R.W., Smith D.G., Murray K.J., Reith A.D., Holder J.C. (2000). Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol., 7, 793–803. 64. Lochhead P.A., Coghlan M., Rice S.Q., Sutherland C. (2001). Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes, 50, 937–946. 65. Eldar-Finkelman H., Schreyer S.A., Shinohara M.M., LeBoeuf R.C., Krebs E.G. (1999). Increased glycogen synthase kinase-3 activity in diabetes- and obesityprone C57BL/6J mice. Diabetes, 48, 1662–1666. 66. Nikoulina S.E., Ciaraldi T.P., Mudaliar S., Mohideen P., Carter L., Henry R.R. (2000). Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes, 49, 263–271. 67. Liberman Z., Eldar-Finkelman H. (2005). Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling. J. Biol. Chem., 280, 4422–4428. 68. Eldar-Finkelman H., Krebs E.G. (1997). Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. Proc. Natl. Acad. Sci. USA, 94, 9660–9664. 69. Pearce N.J., Arch J.R., Clapham J.C., Coghlan M.P., Corcoran S.L., Lister C.A., Llano A., Moore G.B., Murphy G.J., Smith S.A., Taylor C.M., Yates J.W., Morrison A.D., Harper A.J., Roxbee-Cox L., Abuin A., Wargent E., Holder J.C. (2004). Development of glucose intolerance in male transgenic mice overexpressing human glycogen synthase kinase-3beta on a muscle-speciﬁc promoter. Metabolism, 53, 1322–1330. 70. Cole A.R., Knebel A., Morrice N.A., Robertson L.A., Irving A.J., Connolly C.N., Sutherland C. (2004). GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons. J. Biol. Chem., 279, 50176–50180. 71. Jiang H., Guo W., Liang X., Rao Y. (2005). Both the establishment and the maintenance of neuronal polarity require active mechanisms: Critical roles of GSK-3beta and its upstream regulators. Cell, 120, 123–135. 72. Yoshimura T., Kawano Y., Arimura N., Kawabata S., Kikuchi A., Kaibuchi K. (2005). GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell, 120, 137–149. 73. Zumbrunn J., Kinoshita K., Hyman A.A., Nathke I.S. (2001). Curr. Biol., 11, 44–49. 74. Maccioni R.B., Munoz J.P., Barbeito L. (2001). The molecular bases of Alzheimer’s disease and other neurodegenerative disorders. Arch. Med. Res., 32, 367–381. 75. Phiel C.J., Wilson C.A., Lee V.M., Klein P.S. (2003). GSK-3alpha regulates production of Alzheimer’s disease amyloid-beta peptides. Nature, 423, 435–439. 76. Carmichael J., Sugars K.L., Bao Y.P., Rubinsztein D.C. (2002). Glycogen synthase kinase-3beta inhibitors prevent cellular polyglutamine toxicity caused by the Huntington’s disease mutation. J. Biol. Chem., 277, 33791–33798.

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77. Garrido J.L., Godoy J.A., Alvarez A., Bronfman M., Inestrosa N.C. (2002). Protein kinase C inhibits amyloid beta peptide neurotoxicity by acting on members of the Wnt pathway. FASEB J., 16, 1982–1984. 78. Perez M., Rojo A.I., Wandosell F., Díaz-Nido J., Avila J. (2003). Prion peptide induces neuronal cell death through a pathway involving glycogen synthase kinase 3. Biochem. J., 372, 129–136. 79. O’Brien W.T., Harper A.D., Jove F., Woodgett J.R., Maretto S., Piccolo S., Klein P.S. (2004). Glycogen synthase kinase-3beta haploinsufﬁciency mimics the behavioral and molecular effects of lithium. J. Neurosci., 24, 6791–6798. 80. Gould T.D., Gray N.A., Manji H.K. (2003). Effects of a glycogen synthase kinase3 inhibitor, lithium, in adenomatous polyposis coli mutant mice. Pharmacol. Res., 48, 49–53. 81. Ring D.B., Johnson K.W., Henriksen E.J., Nuss J.M., Goff D., Kinnick T.R., Ma S.T., Reeder J.W., Samuels I., Slabiak T., Wagman A.S., Hammond M.E., Harrison S.D. (2003). Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes, 52, 588–595. 82. Cline G.W., Johnson K., Regittnig W., Perret P., Tozzo E., Xiao L., Damico C., Shulman G.I. (2002). Effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in Zucker diabetic fatty (fa/fa) rats. Diabetes, 51, 2903–291

2 GLYCOGEN SYNTHASE KINASE 3b (GSK-3b) A KEY SIGNALING ENZYME: A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE Nibaldo C. Inestrosa, Ginny Farías, and Marcela Colombres Centro de Regulación Celular y Patología “Joaquín V. Luco” (CRCP), MIFAB, Facultad de Ciencias Biológicas, Pontiﬁ cia Universidad Católica de Chile

Contents 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9

Introduction Role of Wnt Signaling and GSK-3 in Development Embryonic Stem Cells Modulation by Wnt Signaling Components Neuronal Polarity and GSK-3β GSK-3β Activation in Neurodegeneration Aβ-AChE Complexes Induce a Loss of Function of the Wnt Pathway GSK-3β as a Clinical Target in Alzheimer’s Disease M1 Muscarinic Receptor Activation Protects from Aβ Toxicity Bifunctional Compound, AChE Inhibitor, and Anti-inﬂammatory Drugs Play a Role in GSK-3β Regulation 2.10 Neuroprotection by PPARγ Activation against Aβ Toxicity 2.11 Concluding Remarks

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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2.1

A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

INTRODUCTION

The Wnt signaling pathway has been described as an essential signaling pathway in cell adhesion and regulation of cell fate determination during animal development [1]. Wnt ligands were identiﬁed by sequence homology with Wnt1 in mouse and Wingless (Wg) in Drosophila, which encoded for secreted signaling glycoproteins. The classical view of the canonical Wnt signaling transduction pathway implies the presence of an extracellular secreted Wnt ligand that interacts with a membrane-associated seven-transmembrane receptor protein called the Frizzled receptor. These glycoproteins form a rather interesting protein family that usually transduces its signal to the cytoplasmic compartment, and activates Disheveled (Dsh; usually by phosphorylation), which in turn inactivates glycogen-synthase-kinase-3β (GSK-3β), a key modulator of this signaling pathway. GSK-3β forms a high-molecular weight complex that includes Axin, the adenomatous poliposis coli (APC) component, and β-catenin [2]. The net result, of the activation of the canonical Wnt signaling, is that the intracellular levels of β-catenin increase, allowing its import to the nucleus where it binds to components of the high-mobility group family of transcription factors called the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) and activates the expression of Wnt target genes, which includes several genes, such as engrailed and cyclin-D1. Conversely, in the absence of a Wnt ligand, the activity of GSK-3β is switched on and phosphorylates β-catenin, which becomes a substrate for the ubiquitin-proteosome-mediated degradation [3]. Consequently β-catenin levels diminish within the cytoplasm, and so the expression of Wnt target genes in the nuclei. So far the Wnt functions include control of tissue patterning, cell fate determination, cell migration, insulin action, cell polarity, circadian rhythm, apoptosis, proliferation, and development of the nervous system.

2.2 ROLE OF WNT SIGNALING AND GSK-3 IN DEVELOPMENT The development of animal organs requires a coordination of cell proliferation and morphogenesis processes. Wnt proteins have been described to regulate early events in the developing nervous system. Wnt3, -3a, -7b, and -8b participate in the development of the forebrain, a region that gives rise to the hippocampus. Ablation of Wnt1 results in severe defects of the midbrain, the cerebellum and the developing spinal cord, while ablation of Wnt3a results in a total loss of the hippocampus. The deletion of the Wnt1 gene also results in loss of the midbrain–hindbrain junction and the consequent loss of dopaminergic (DA) neurons [4]. In fact Wnts are involved in the acquisition of a DA phenotype in the developing ventral mesencephalon. Wnt1 mainly regulates the proliferation of neural precursors while Wnt5a is involved in the conversion of precursors positive for nuclear receptor-related factor 1 into DA neurons. Wnts are palmitoylated glycoproteins, poorly soluble ligands, prop-

EMBRYONIC STEM CELLS MODULATION

27

erty that limitis their potential use in a clinical set up. One proposed way to bypass this limitation could be the development and application of drugs that modulate Wnt signaling pathways. In this aspect, chemical inhibitors of GSK3β, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in ventral mesencephalon precursor cultures [5]. In addition the GSK-3β-speciﬁc inhibitor kenpaullone increased the size of the DA neuron population tyrosine hydroxylase positive neurons, mimicking an effect of Wnt’s. Furthermore it was shown that GSK-3β inhibitors stabilized βcatenin in ventral mesencephalic precursors resulting in increased DA differentiation. These compounds have been suggested to be used in the improvement of stem cell therapy approaches in neurodegenerative diseases. In Drosophila the Wingless signaling pathway controls the formation of the antero-posterior boundary of the Imaginal discs, and the homologous Wnt pathways in vertebrates are essential for development of many organs [6]. These pathways also control cell proliferation in respective target tissues. In Drosophila the Wingless pathway requires armadillo (homologue of βcatenin), while the GSK-3 homologue shaggy (also known as zeste-white 3) negatively regulates the pathway and thus proliferation of the target cells. In fact Drosophila shaggy plays a critical role during Drosophila neurogenesis. The ectopic expression of Wingless leads to induction of ectopic structures and general overproliferation of the surrounding cells [7]. Interestingly, Wnt1 (a Wg homologue in mice) has been implicated in development of vertebrate brain [4]. During Xenopus embryonic development GSK-3 acts as a negative regulator of dorsal axis formation [8]. Inhibition of GSK-3 activity leads to stabilization of β-catenin and expression of target dorsalizing genes [9,10]. GSK-3 promotes β-catenin phosphorylation, which stimulates its degradation by the ubiquitin-proteasome system [11]. Maternally expressed β-catenin is essential for dorsal determination and is localized to the future dorsal side of the embryo just after egg fertilization [12,13]. Thus meiotic maturation can be considered as a preparation for asymmetric localization of β-catenin and dorsal axis formation. Interestingly insulin, as well as progesterone, can promote meiotic maturation of Xenopus oocytes.

2.3 EMBRYONIC STEM CELLS MODULATION BY WNT SIGNALING COMPONENTS Embryonic stem cells (ESC) are self-renewing cells found in the inner cell mass of the blastocyst stage embryo and characterized by maintaining pluripotency. These cells can differentiate in vivo and in vitro into all cell lineages of the adult animal. Stem cell self-renewal and progenitor differentiation is regulated by the specialized micro-environment in which these cells reside. Human-ESC could be used to generate speciﬁc types of tissues for cell

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A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

replacement therapy in diseases like diabetes mellitus. Little is known, however, about the signaling pathways that govern ESCs [14]. Wnt proteins are able to induce the proliferation in different types of stem cells such as embryonic, intestinal, skin, and haematopoietic stem cells. In the central nervous system, Wnt’s act mitogenically on progenitor cells, and activation of β-catenin leads to ampliﬁcation of the neural progenitor pool. Interestingly the canonical Wnt pathway components are detected in undifferentiated ESCs. The function of the Wnt signaling pathway in the central nervous system (CNS) has been extensively studied during neuronal development; however, in adult CNS it is less characterized. It has been described that an increase in Wnt7a signaling mediates the maturation of synaptic contacts between cerebellar granule cells and mossy ﬁbers in the cerebellum cortex in vivo by remodeling axonal microtubules, without the need of transcriptional activity of β-catenin. Other studies have shown that incomplete Wnt signaling occurs during depolarization-induced β-catenin-mediated dendritic arborization. In cultured hippocampal neurons, Wnt ligands with β-catenin promoting activity are modulated by depolarizating-inducing stimulus and hence βcatenin stabilization regulation and increased N-cadherin levels mediate dendritic morphogenesis. The Wnt signaling has been implicated in early stages of neural crest development, such as neural crest induction and melanocyte formation. Neural crest cells generate most structures of the peripheral nervous system and nonneural tissues such as cells in the the heart, craniofacial bone and cartilage, connective tissue, and melanocytes of the skin [15]. In neural crest stem cells (NCSCs), the genetic ablation of β-catenin results in lack of melanocytes and sensory neural cells in dorsal root ganglia. In fact NCSCs without β-catenin emigrate and proliferate normally but are unable to acquire a sensory neuronal fate. Constitutive expression of β-catenin in neural stem/progenitor cells results in an expansion of the entire neural tube [16,17], supporting a role of β-catenin in progenitor proliferation. On the other hand, effects of ablation of wnt1 and wnt3 suggest a role of Wnt signaling in expansion of dorsal neural tube cells, including the premigratory neural crest. Similar to other growth factors that promote alternative fates, Wnt/β-catenin induces sensory neurogenesis by acting instructively on embryonic NCSCs [18]. Mutations in other Wnt components like APC, which are associated with increased tumor incidence and increased intracellular levels of β-catenin, interfere with embryonic stem cell differentiation into the three germ layers. Interestingly it has been shown that Wnt ligands promote differentiation of neural stem cells in the neocortex at E11.5 at the expense of neural stem cell expansion. In another study, in which neural stem cells derived from the telencephalon were cultured as neurospheres and the Wnt signaling was activated, it was reported that Wnt3a inhibits the maintenance of neural stem cells and promotes the differentiation into neuronal and astrocyte lineages. Interestingly a recently discovered, cell-permeable GSK-3 inhibitor known as derivative, 6bromoindirubin-3″-oxime (BIO), facilitates the maintenance phenotype and

GSK-3β ACTIVATION IN NEURODEGENERATION

29

the sustained expression of pluripotent state-speciﬁc transcription factors in mouse and human ESCs [19]. The indirubin family of GSK-3β inhibitors was initially isolated from Chinese medicines used against chronic diseases such as chronic myelocytic leukaemia [20]. Indirubins derive from a nonenzymatic dimerization of isatin and indoxyl, two precursors found in over 200 species of indigo-producing plants [21].

2.4

NEURONAL POLARITY AND GSK-3b

The neuron is one of the most highly polarized cells characterized by two structurally and functionally distinct parts, an axon (from an elongation of one of the immature neurites) and dendrites. Dendrites usually receive signals, while the axon usually sends signals. Neuronal polarity is essential for information-processing events in a unidirectional form. The mechanisms implicated on how neuronal polarity is controlled still remain not well understood. It has been shown that GSK-3β plays a role in establishing and maintaining the axon-dendrite polarity. Interestingly, after the transition from st. 2 nonpolarized neurons to st. 3 polarized neurons, GSK-3β activity is higher in dendrites than in axons, which is critical for determining axon-dendrite polarity. A protein known as collapsin response mediator protein-2 (CRMP-2) has been described as crucial for axon outgrowth and determination of the fate of the axon and dendrites. CRMP-2 is therefore essential in the establishment and maintenance of neuronal polarity. Further it has been shown that GSK-3β phosphorylates CRMP-2 and inactivates its activity, participating in neuronal polarization through CRMP-2. Also NT-3 and brain-derived neurotrophic factor were shown to inhibit GSK-3β via PKB, reducing the phosphorylated levels of CRMP-2 at Thr514 and thus leading to axon elongation and branching [22]. Phosphorylation of CRMP-2 at Ser518 and Ser522 is implicated in the formation of degenerating neurites in Alzheimer’s disease (AD).

2.5

GSK-3b ACTIVATION IN NEURODEGENERATION

The Wnt signaling pathway has been related to neurodegenerative disorders such as autism, schizophrenia, and AD. Active GSK-3β has been found in brains staged for AD neuroﬁbrillary changes [23], and a decrease in β-catenin levels and an increase in Tau hyperphosphorylation. Also neurodegeneration and spatial learning deﬁcit have been observed in GSK-3β conditional transgenic mice [24,25]. In addition previous studies had shown that β-catenin levels are reduced in AD patients carrying PS-1-inherited mutations [26]. In fact several studies have shown that familial AD-linked presenilin (PS) proteins form multi-protein complexes with the cell adhesion/Wnt signaling β-catenin protein, α-catenin, and GSK-3β. β-Catenin and its homo-

30

A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

logue γ-catenin, commonly named plakoglobin, were initially described as intracellular proteins located near the cell surface and linked to cadherins. Cadherins directly interact with β-catenin or γ-catenin, which binds to αcatenin. β-Catenin is found in four cellular locations: (1) at the plasma membrane, interacting with cadherins and through α-catenin interacts with the cytoskeleton, (2) the presynaptic nerve terminals, (3) in the cytoplasm (free or forming a complex with APC protein), and (4) in in the nucleus. The α-T catenin gene has been mapped in a trait locus on chromosome 10 in late-onset AD. α-T Catenin interacts with β-catenin, which itself interacts with PS-1 making it an interesting functional candidate. Thus mutations in α-catenins may lead to instability of the β-catenin-PS complex causing neuronal increased production of Aβ. Early studies in our laboratory suggested a relationship between Aβinduced neurotoxicity and lower cytoplasmatic levels of β-catenin. On the other hand, inhibition of GSK-3β by lithium protects rat neurons from Aβinduced damage. These evidences led to the proposal that a sustained loss of Wnt signaling function may be involved in the Aβ dependent neurodegeneration observed in AD. It has been demonstrated that Wnt3a conditioned media prevents neuronal cell death in Aβ-treated rat hippocampal cultures. Frzb-1, a secreted Wnt antagonist protein, was able to reverse the Wnt3a neuroprotective effect on cell survival against Aβ toxicity. Furthermore, when hippocampal neurons were exposed to Aβ-ﬁbrils, a decrease in their cytosolic β-catenin level was observed; this effect was also reversed when Wnt3a ligand was co-incubated with Frzb-1. These results suggest that Wnt/β-catenin signaling plays an important role in neuroprotection against Aβ toxicity, and its loss of function may play a crucial role in triggering the neurodegeneration observed in AD. Our ﬁ ndings are in agreement with observations that suggest that Aβ induces aberrant cell cycle activation in neurons through mitogenic Wnt signaling and later by Wnt signaling downregulation by Aβ through induction of the Wnt antagonist, dickkopf. Aβ neurotoxicity has been associated with neuronal shrinkage and dendritic dystrophy similar to those observed in AD neurons that develop neuroﬁbrillar pathology. In primary cultures of hippocampal and cortical neurons exposed to Aβ neurotoxicity, an increased activation of GSK-3β, the hyperphosphorylation of Tau proteins, and the loss of the microtubule network have all been observed [27,28]. Aβ decreases the number of neurites on hippocampal primary cultures and Wnt ligand is able to prevent this effect. Exposure of neurons in culture to Aβ induces apoptosis and promotes Tau hyperphosphorylation, through GSK-3β activity. The inactivation of GSK-3β may result from the action of PKC, leading to two main consequences: reduced phosphorylation of Tau protein and reduced degradation and subsequent accumulation of cytoplasmatic β-catenin. Moreover PKC isoenzymes are degraded in a differential manner upon Aβ exposure. The modulation of PKC in fact affects Aβ neurotoxicity, as the activation of this enzyme by phorbol 12-myristate 13-acetate increases cell viability of rat hippocampal neurons

31

GSK-3β ACTIVATION IN NEURODEGENERATION

and neuroprotection toward Aβ ﬁbrils. PKC inhibits GSK-3β through serine 9, preventing the cytoplasmatic β-catenin degradation and thus activating the transcription of Wnt target genes such as engrailed and cyclin-D1. However, the inhibition of Ca2+ -dependent PKC isoforms activated GSK-3β and showed no protection against Aβ induced neurotoxicity. Wnt3a and lithium mimicked PKC activation. The regulation of some components of the Wnt signaling pathway by Ca2+ -dependent PKC isoforms, may be important in controlling the neurotoxic process induced by Aβ. A loss of signaling through the β-catenin-Tcf pathway increases neuronal vulnerability to apoptosis induced by Aβ [26], and possible defects in Wnt signaling could contribute to the pathogenesis of AD [29–31]. Although the mechanism by which the Wnt signaling plays a role in AD pathology remains unclear, recent evidence show that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components (Fig. 2.1) [32,33].

A

Ab Present

B

Ab Absent

Aβ Aβ

Wnt

Aβ

Fzd

Fzd

LiCl

GSK-3b active

b-catenin P (degradation)

Wnt target genes transcription OFF nucleus

GSK-3b inactive

b -catenin b -catenin b -catenin (stabilization)

Wnt target genes transcription ON nucleus

Figure 2.1 The Wnt signaling pathway is a target for Aβ toxicity. (A) Effect of Aβ on Wnt signaling molecules. Aβ in hippocampal neurons impairs cell survival, increases GSK-3β, decreases cytoplasmic β-catenin and decreases the expression of Wnt target genes. Lithium can attenuate Aβ neurotoxicity by recovering β-catenin levels and Wnt target gene expression. (B) Wnt signaling pathway activation. Wnt ligands act through Frizzled receptors (Fz) at the plasma membrane leading to the inactivation of GSK-3β. As a consequence β-catenin is stabilized and accumulated on the cytoplasm, where it can translocate to the nucleus and promote the transcription of Wnt target genes. See color plates.

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A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

GSK-3β has been reported to facilitate apoptosis [34,35], and in previous studies we showed that lithium and PKC activators prevent the neuronal impairment induced by Aβ-toxicity through Wnt signaling by inhibition of GSK-3β activity [33,36]. It has also been described that GSK-3α regulates the Aβ peptide production. Therapeutic concentrations of lithium (1–2 mM) interfere with APP cleavage at the γ-secretase step and blocks the accumulation of Aβ peptides in brains of mice overproducing Aβ. Therefore compounds that inhibit GSK3α could lead to the reduction of both the formation of amyloid plaques and neuroﬁbrillary tangles, two hallmarks of AD.

2.6 Ab-ACHE COMPLEXES INDUCE A LOSS OF FUNCTION OF THE WNT PATHWAY Previously it was shown that 1 to 10 μM of Aβ ﬁbrils and Aβ-AChE complexes induce neuronal cell death in neuronal cell lines and primary cultures of rat hippocampal neurons and chick retina cells. The Aβ-AChE complexes neurotoxicity showed more damage than Aβ ﬁbrils alone, an effect that is dependent on the AChE concentration associated to the ﬁbrils. It was then examined whether lithium could act as a neuroprotective factor against the damage generated by the Aβ-AChE complexes. Primary cultures of rat hippocampal neurons exposed to these complexes showed clear morphological alterations including somatic shrinkage plus severe axonal and dendritic dystrophy and a signiﬁcant impairment of neuronal cell activity. Almost all these changes were prevented by co-incubation with 100 μM lithium. β-catenin levels have been analyzed in hippocampal neurons treated with Aβ. These studies reveal a decrease in cytoplasmic β-catenin levels after 6 to 8 hours of treatment. However, this effect occurs as early as 2 hours of incubation when the Aβ-AChE complexes were used. Both effects were reverted to control levels with lithium.

2.7 GSK-3b AS A CLINICAL TARGET IN ALZHEIMER’S DISEASE Neuroprotective targets for AD are still elusive because knowledge of the molecular events that underlie Aβ neurodegeneration and apoptosis is not complete [37,38]. The emerging role of Wnt signaling as a therapeutic target for treatment of AD led us to evaluate potential pathways that interact with the Wnt signaling. Since GSK-3β activation seems to be a target of Aβinduced apoptosis, different GSK-3β target drugs have been evaluated to avoid the Aβ neurotoxicity by cross-talk with the Wnt signaling pathway. These studies are expanding the novel approaches for clinical treatment and therapies.

A ROLE IN GSK-3β REGULATION

33

2.8 M1 MUSCARINIC RECEPTOR ACTIVATION PROTECTS FROM Ab TOXICITY In AD a degeneration of presynaptic cholinergic neurons that ascend from the basal forebrain to cortical and hippocampal areas has been observed [39,40]. The M1 muscarinic receptor is expressed in the cerebral cortex and hippocampus, and its major role is in cognitive processing including shortterm memory [41,42]. In relation to AD, it is well known that M1 agonists increase the nonamyloidogenic processing of the amyloid precursor protein (APP), reducing Aβ production [43–35] and also Tau phosphorylation [46,47]. Although the precise mechanism by which M1 muscarinic receptor activation may promote neuroprotection remain unclear; a possibility is through inhibition of GSK-3β. It has been demonstrated that the muscarinic receptor induced activation of protein kinase C (PKC) is mediated through G protein signaling pathways. Interestingly it has been shown that PKC protects from apoptosis induced by Aβ [36,48]. We have found that M1 muscarinic receptor activation protects from Aβ toxicity through Wnt signaling activation [49]. Hippocampal neurons exposed to Aβ toxicity induced the activation of GSK-3β as evidenced by an increase in the activity of GSK-3β, which was prevented by the activation of m1 muscarinic receptor by the speciﬁc agonist, AF267B (Fig. 2.2). In addition the M1 muscarinic receptor’s activation induces the phosphorylation/inactivation of GSK-3β in cortical neuronal cultures from transgenic mice that overexpress GSK-3β. GSK-3β is negatively regulated by phosphorylation at Ser9 [50,51]. Aβ treatment in transgenic mice that overexpress GSK-3β shows decreased levels of Ser9 phosphorylation; thus GSK3β is activated. M1 agonist treatments decrease GSK-3β activity through an increase in Ser9 phosphorylation [49]. The inactivation of GSK-3β may occur through PKC, since PKC can inactivate GSK-3β through Ser9 phosphorylation [36], and M1 mAChR activation is capable to activate PKC via G protein [44]. In this manner Ser9 phosphorylation/inactivation of GSK-3β by M1 mAChR stimulation is probably mediated by a mechanism that involves PKC, since a PKC inhibitor blocked M1 muscarinic receptor activation-induced Ser9 phosphorylation (Fig. 2.3) [49]. Thus the M1 muscarinic activation and Wnt signaling pathways interact, leading to neuroprotection against Aβ neurotoxicity.

2.9 BIFUNCTIONAL COMPOUND, ACHE INHIBITOR, AND ANTI-INFLAMMATORY DRUGS PLAY A ROLE IN GSK-3b REGULATION The cholinergic dysfunction observed in AD patients has aimed to understand the relationship between Aβ neurotoxicity and cholinergic function.

34

A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

A

CH3 CH3

O C

OH

CH3

O

CH3

O Pyridostigmine (PO) O

O C

Br-

O

CH3

OCN CH3

N+

CH3

CH3

N

N

CH3 Ibuprofen (IBU) CH3

CH3

CH3

CH3

(±)IBU-PO

B

C

Rosiglitazone AF267B

O

D CH3 H3C HO

O O

NH

S O

CH3 CH3

Troglitazone

Figure 2.2 (A) IBU-PO is a bifunctional compound that combines ibuprofen (IBU), a nonsteroidal anti-inﬂammatory drug (NSAID) with pyridostigmine (PO), a cholinesterase inhibitor (ChEI). (B) The highly rigid compound AF267B is a selective M1 mAChR agonist with functional selectivity preserved both at the level of the M1 mAChR as well as along select signal transduction pathways. (C and D) Rosiglitazone and Troglitazone are thiazolidinedione drugs acting as agonists of PPARγ.

AChE has been found to co-localize with Aβ deposits [52] and promotes the assembly of Aβ into amyloid ﬁbrils forming Aβ-AChE complexes that are more toxic than amyloid ﬁbrils when exposed to rat hippocampal neurons [53–55]; recently we have found that Aβ-AChE complexes involved a loss of Wnt signaling [56]. These observations suggest that AChE has a role in the pathogenesis of AD. It has been observed that AChE is present in senile plaques in brains from AD patients [52,57]. Moreover AChE increases Aβ aggregation in Alzheimer’s ﬁbrils [54] and the Aβ-AChE complexes induce the death of neurons in vitro more dramatically than Aβ ﬁbrils alone [53,55,58]. Thus several cholinesterase inhibitors (ChEI) have been developed and approved for the treatment of AD patients [40,59,60].

35

A ROLE IN GSK-3β REGULATION

Ab

? ?

Ab Ab AChE Ab

AChE-Inhibitor M1 mAChR agonist

Anti-diabetic drugs

ACh

Wnt

ACh

Fzd-R

PKC

p-Ser 9

GSK-3b

P

b-catenin

G

PPARg Ub

Cytosol Nucleus

b-catenin Wnt target genes Tcf/LEF

Figure 2.3 Cross-talk with other cell-signaling pathways that lead to neuroprotection against Aβ through GSK-3β activity inhibition. In the presence of Aβ- or Aβ-AChE toxicity GSK-3β is activated, inducing β-catenin phosphorylation and degradation, turning off the transcription of Wnt target genes. M1 muscarinic agonists activate the m1 muscarinic receptor and induce a GSK-3β inhibition by Ser9 phosphorylation in a PKC-dependent way. In other scenario, diabetic drugs through PPARγ activation also may lead to GSK-3β inhibition by a mechanism not well understood. On the other hand, AChE inhibitor may inhibit GSK-3β through the cholinergic system by the activation of the acetylcholine receptor as m1 mAChR. In a pathological scenario where the Wnt signaling could be affected by Aβ and Aβ-AChE neurotoxicity, the treatment with different drugs that have a cross-talk with the Wnt signaling may to lead to neuroprotection in AD. See color plates.

On the other hand, the use of nonsteroidal anti-inﬂammatory drugs (NSAIDs) has been observed in a clinical study that reduce ﬁvefold the risk of AD [61]. The NSAIDs have been proposed to act that inhibiting the secretases that cleave the amyloid precursor protein (APP) in the amyloidogenic pathway to render Aβ. Moreover NSAIDs dramatically reduce the secretion of Aβ1-42 in cells in vitro [37,62–64]. A bifunctional compound that includes a ChEI and an anti-inﬂammatory drug, IBU-PO (Table 2.1), protects hippocampal neurons from Aβneuro-

c. and d. Rosiglitazone and Troglitazone are thiazolidinedione drugs acting as agonists of PPARγ.

b. The highly rigid compound AF267B is a selective M1 mAChR agonist with functional selectivity preserved both at the level of the M1 mAChR as well as along select signal transduction pathways.

a. IBU-PO is a bifunctional compound that combines ibuprofen (IBU), a nonsteroidal anti-inﬂammatory drug (NSAID) with pyridostigmine (PO), a cholinesterase inhibitor (ChEI).

TABLE 2.1

CH3

CH3

C

O

CH3 C

O

OH

Rosiglitazone

Ibuprofen (IBU) CH3

CH3

CH3

O

HO

H3C

(±)IBU-PO

CH3

CH3

CH3

CH3

N

O O

N

O

N+

CH3

O

O

O

CH3

CH3

NH

CH3

Troglitazone

AF267B

OCN

O

S

CH3

Pyridostigmine (PO)

Br-

CH3

36 A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

NEUROPROTECTION BY PPARγ ACTIVATION AGAINST Aβ TOXICITY

37

toxicity, increases the viability of Aβ-challenged hippocampal neurons, and enhances neurite growth. The protection observed is the result of the Wnt signaling activation, since the increased in the activity of GSK-3β induced by Aβ is downregulated by co-treatment with IBU-PO. In addition this downregulation is through induction of Ser9 phosphorylation. Transgenic mice that overexpress GSK-3β show low levels of Ser9 phosphorylation, and the IBU-PO treatment induces an increase in this phosphorylation (Fig. 2.3) [65]. Compounds such as IBU-PO that mimic the activation of the Wnt signaling pathway could eventually rescue neurons from cytotoxicity, through GSK-3β inhibition, which has potential beneﬁt for the treatment of AD patients.

2.10 NEUROPROTECTION BY PPARg ACTIVATION AGAINST Ab TOXICITY Peroxisome proliferator–activated receptor (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that bind to the peroxisome proliferators-responsive element as a heterodimer with the 9-cis retinoic acid receptor [66]. PPARγ participates in differentiation, insulin sensitivity, type 2 diabetes, arteriosclerosis, and cancer [67,68]. In particular, PPARγ is a critical regulator of adipocyte differentiation [67], a process also dependent on the Wnt signaling pathway [69]. The presence of PPARγ in the brain has been difﬁcult to detect but recently has been shown in hippocampus and cortex [70]. PPARγ protein levels change in the temporal cortex of AD patients suggested that it might play a role in AD pathophysiology [71]. In our laboratory we determined that PPARγ is expressed in rat hippocampal neurons culture, and interestingly the PPARγ activation with the agonist and anti-diabetic thiazolidinedione drugs troglitazone and rosiglitazone (Fig. 2.2) prevent Aβ neurotoxicity [72]. In addition this effect of PPARγ activation in the neuroprotection from the Aβ toxicity correlates with the modulation of the Wnt pathway, where an inhibition of GSK-3β activity is observed with a subsequently β-catenin stabilization (Fig. 2.3) [72]. Recent studies suggest that treatment of insulin resistance with a PPARγ agonist retards the development of AD [73], and recent studies have shown that some anti-inﬂammatory drugs that are PPARγ agonists have neuroprotective actions in different animal models of neurodegeneration [74,75]. Accordingly anti-diabetic thiazolidinedione drugs have been shown to have a potent insulin-sensitizing action [76] that might be mediated through PPARγ-mediated inhibition of GSK-3β [77]. These ﬁ ndings suggest that the PPARγ pathway cross-talk with the Wnt signaling pathway underlies the neuroprotection from Aβ toxicity.

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2.11

A DEVELOPMENTAL NEUROBIOLOGICAL PERSPECTIVE

CONCLUDING REMARKS

The potential uses of GSK-3 in development and embryonic stem cell modulation as well as its targets in the therapy of neurodegenerative disorders have been discussed. Compounds that inhibit the activity of GSK-3 could lead to the reduction of neuropathological factors implied in AD. The Wnt signaling pathway has been implicated in several cellular functions. The cross-talk of the Wnt signaling pathway with other cellular pathways is opening new possibilities of therapy that include blocking of the pivotal role of GSK-3. ACKNOWLEDGMENTS This work was supported by a grant FONDAP-Biomedicine N° 13980001, from the Millennium Institute for Fundamental and Applied and Biology (MIFAB) and a pre-doctoral CONICYT Fellowship to MC and GF. REFERENCES 1. Patapoutian A., Reichardt L.F. (2000). Roles of Wnt proteins in neural development and maintenance. Curr. Opin. Neurobiol., 10, 392–399. 2. Chen R.H., Ding W.V., McCormick F. (2000). Wnt signaling to β-catenin involves two interactive components: Glycogen synthase kinase-3β inhibition and activation of protein kinase C. J. Biol. Chem., 275, 17894–17899. 3. Yost C., Torres M., Miller J.R., Huang E., Kimelman D., Moon R.T. (1996). The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev., 10, 144314–144354. 4. McMahon A.P., Bradley A. (1990). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell, 62, 1073–1085. 5. Castelo-Branco G., Rawal N., Arenas E. (2004). GSK-3β inhibition/β-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J. Cell Sci., 117, 5731–5737. 6. Cadigan K.M., Nusse R. (1997). Wnt signaling: A common theme in animal development. Genes Dev., 11, 3286–3305. 7. Díaz-Benjumea F.J., Cohen S.M. (1995). Serrate signals through notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Drosophila wing. Development, 121, 4215–4225. 8. Dominguez I., Itoh K., Sokol S.Y. (1995). Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos. Proc. Natl. Acad. Sci. USA, 92, 8498–8502. 9. Wylie C., Kofron M., Payne C., Anderson R., Hosobuchi M., Joseph E., Heasman J. (1996). Maternal β-catenin establishes a “dorsal signal” in early Xenopus embryos. Development, 122, 2987–2996.

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3 ROLE OF GSK-3/SHAGGY IN NEURONAL CELL BIOLOGY Diana Simón, Olga Varea, Juan José Garrido, and Francisco Wandosell Centro de Biología Molecular “Severo Ochoa,” Madrid

Contents 3.1 Cytoskeletal Remodeling Associated with Neurite Extension 3.2 GSK-3 in Neuronal Development 3.2.1 Distribution of GSK-3 in Neurons 3.3 Role of GSK-3 in Neuronal Morphogenesis 3.3.1 Role of GSK-3 in Determination and Maintenance of Neuronal Polarity 3.4 How Is GSK-3 Activity Controlled? 3.5 Pathways Controlling GSK-3 Activity in Neurons 3.5.1 Insulin/IGF1 3.5.2 Wnt Signaling 3.5.3 Estradiol Signaling 3.5.4 NGF Signaling 3.5.5 Reelin Pathway 3.5.6 LPA as Regulator of Neuron Morphology through Inﬂuence on the Cytoskeleton 3.6 Signiﬁcance of GSK-3 Activation 3.7 Putative Physiopathological Implications of GSK-3 Activation 3.8 Concluding Remarks

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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3.1 CYTOSKELETAL REMODELING ASSOCIATED WITH NEURITE EXTENSION The nervous system is a highly ordered network of neural connections. These connections are ﬁ rst established during development, and they are based on the capacity of neuroblasts to extend cytoplasmic processes known as neurites. During this differentiation process, referred to as neuritogenesis, growing neurites follow long and stereotypic routes to reach their speciﬁc targets. As a result of these processes neurons become fully differentiated, establishing one of the clearest examples of the linkage between morphology and function underwritten by the modiﬁcations undertaken by the cytoskeleton. Moreover the morphology of these neurons is based on the capacity of the cytoskeleton to assembly and disassembly in response to extracellular signals. During neurite outgrowth microﬁ laments (MFs) and microtubules (MTs) constitute the major cytoskeletal components of neurons. Both these cytoskeletal elements are present in the neuroblast, and they play a key role in the ﬁ rst steps of neurite extension. The growth of the neurite is guided and controlled by the growth cone that forms at its tip. The peripheral cytoplasmic domain of the growth cone is ﬁlled with MFs, made up of ﬁ lamentous actin (F-actin) and a complex set of actin binding proteins (ABPs). In this peripheral region two different structures may be distinguished, the ﬁ lopodia that corresponds to the ﬂattened leading edge of the growth cone and the lamellipodia in which actin forms a dense mesh. Even though the molecular mechanisms that control actin dynamics are not completely understood [1], it is clear that some accessory proteins, such as the small GTPases of the Rho family, play a central role in the changes undertaken by the actin cytoskeleton in the growth cone. MTs are the most prominent components of the neurite shaft and the central region of the growth cone. They are essential for the formation and the maintenance of neurite extension, as their polymerization and subsequent stabilization leads to the net extension and consolidation of the growing neurite. Microtubules are polymers of α/β-tubulin heterodimers and tubulin polymerization is a highly dynamic process, implying that MTs are constantly in transit between phases of growth and shrinkage (“MT dynamic instability”). Two different groups of tubulin-binding proteins have been described: microtubule-interacting proteins (MIPs) and microtubule-associated proteins (MAPs). While MAPs increase the intrinsic polymerization capacity of tubulin, controlling their dynamics, and they contribute to the organization of the microtubule cytoskeleton, MIPs do not. Many of the already characterized MAPs are highly expressed in the nervous system, and some of them, such as MAP1B, Tau, MAP2, and CRMP2, are even neuron speciﬁc. Indeed, some are predominantly localized in axons (e.g., Tau), whereas others such MAP2 are almost exclusively conﬁ ned to somatodendritic compartments [2]. The capacity of each MAP to stabilize MTs appears to be different, and as a general rule, this is regulated by its phosphorylation state. Phosphoryla-

GSK-3 IN NEURONAL DEVELOPMENT

47

tion of MAPs plays a key role during axonal outgrowth, and the subsequent stabilization and consolidation of MTs in the neurite should be seen as a graduated process that progresses in a proximal to distal direction. Accordingly, the MTs that are located more proximal to the neuronal cell body contain MAPs that are phosphorylated to a lesser extent and they are consequently more stable. In contrast, in the MTs located more distally, in or near to the growth cone, the MAPs are more heavily phosphorylated and are therefore more dynamic. For this reason the regulation of speciﬁc kinases and phosphatases throughout the development of the nervous system in vivo is essential to control neurite extension. In this respect there is a large body of data indicating that glycogen synthase kinase-3 (GSK-3) plays a essential role in neuronal morphogenesis and neuronal survival.

3.2 GSK-3 IN NEURONAL DEVELOPMENT It is generally accepted that in many cell types, GSK-3 plays an important role in several critical physiological processes including the cell cycle, apoptosis, and development. Indeed, in terms of neuronal physiology GSK-3 has been implicated in pathways that control morphogenesis, synaptogenesis [3] or survival [4], and it has even been associated with pathological processes such as Alzheimer’s disease (AD) [5,6]. GSK-3 was initially identiﬁed as one of the serine/threonine protein kinases that phosphorylates and inhibits glycogen synthase. However, a plethora of different substrates have since been identiﬁed, among which metabolic proteins [7], cytoskeletal proteins, and even signal transduction and transcription factors [8] can be found. At least three isoforms of GSK-3 have been shown to exist, the alpha (α), beta (β1 and β2) isoforms encoded by two different genes [9,10]. Furthermore GSK-3 appears to be widely distributed, although its level of expression may vary between different tissues and cell types or even in the same tissue at different developmental stages. For example, the level of GSK-3 is high in the brain at E18, and it reaches a peaked at P8, after which it decreases [11]. While this pattern suggests that GSK-3 is involved in neuronal development, the possible function of each isoform in these processes has not been fully elucidated.

3.2.1

Distribution of GSK-3 in Neurons

Ground-breaking immunohistochemical studies showed that GSK-3 is mainly present in neurons, and interestingly it was found in growing axons [11]. However, in mature tissue it appears that GSK-3 becomes restricted to the gray matter. These studies showed that the developmental proﬁ les of GSK-3α and GSK-3β expression are different and in particular, that the β isoform is downregulated after birth.

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More recent data has shown that GSK-3α. and β are expressed in many neuronal compartments and in primary neurons they can be found in the axon, dendrite, or even in the nucleus [12] (see Fig. 3.1). It has also been suggested that the nuclear translocation of GSK-3 may be associated with apoptotic processes however, in other paradigms of neuronal death this has been not been ratiﬁed (D. Simon, unpublished data). It is notable that a proportion

1

4

2

5

3

6

Figure 3.1 Serial sections (n = 6) of confocal images of differentiated cortical neurons. Cortical primary neurons were cultured for 30 h in a serum-free deﬁ ned medium (NB-B27). Then neurons were ﬁ xed and inmnunostained with antibody against pSer-GSK-3(α/β).

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ROLE OF GSK-3 IN NEURONAL MORPHOGENESIS

of GSK-3, mostly the β isoform, has been reported to be associated with Factin in the growth cone. This pool appears to respond rapidly to external signals such as Semaphorins, being modiﬁed by phosphorylation and/or relocated in the growth cone [13].

3.3

ROLE OF GSK-3 IN NEURONAL MORPHOGENESIS

Different approaches have been adopted in order to deﬁ ne the precise role of GSK-3 in neurons. Unfortunately, mutant mice lacking GSK-3β have proved to be uninformative, since disruption of the murine GSK-3 beta gene provokes embryonic lethality due to severe liver degeneration at midgestation [14]. Nevertheless, the recent analysis of hippocampal neurons in cell culture has provided evidence for a novel role for GSK-3 in axon speciﬁcation.

GF′s

ApoE R2 VLDLR

Akt

p110

p85

p110

PIP3 IRS

LPA

Wnt LRP

TrK′s

IGF-R

Reelin

p85

Gα12 Gα13

GSK-3

APC GSK3-b

RhoA

fyn Akt

PSer

Dsh

Axin

ILK

ER

ER

mDab E2

(?)

Tk(?) PTyr

GSK-3 PSer

PTyr

GSK-3

β-Catenin

PROTEASOME

Figure 3.2 Some pathways that modulate the kinase activity of GSK-3. These pathways may be classiﬁed in two main groups. First are the pathways that act through the inhibition of GSK-3 by increasing Ser9/21 phosphorylatio, such as those triggered by Insulin/IGF-1 (IGF1) or by some growth factors (GFs) or by estrogen (E2). However, so far it is still not clear how GSK-3 is inhibited by the action of Wnt in these Wnt pathways. Second are the class of pathways in which GSK-3 is activated by LPA or Reelin. In these systems the precise molecular mechanism of how GSK-3 is activated still has to be deﬁ ned. See color plates.

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3.3.1 Role of GSK-3 in Determination and Maintenance of Neuronal Polarity A new role for GSK-3 has recently been proposed following the application of two complementary approaches, one taking advantage of different pharmacological inhibitors of GSK-3 and the other using sRNAi against the α. and β isoforms of GSK-3 [15,16]. However, contradictory results have been obtained indicating that either the inhibition or the activation is required to the initiate axon speciﬁcation [15]. The ﬁ rst of these reports indicated that the activity of GSK-3β may regulate a complex in which PAR3, APC, and KIF3A are essential to establish axonal polarity. Indeed, it was proposed that PAR3 polarizes neurons through a mechanism mediated by both APC and kinesin, and that blocking GSK-3 activity inhibits axonal elongation. However, a very distinct scenario was presented in a second report where the inhibition of GSK-3 produced the sprouting of multiple axons in a single neuron, while constitutively active GSK-3β. appeared to inhibit axon formation [16]. To our surprise, both reports used the same inhibitors although the range of concentrations used to produce the effects observed differed between the two. Perhaps the different concentrations used explain the contrasting effects observed. What is apparent is that more work must to be done to clarify the role of GSK-3 in the crucial process of axon morphogenesis. The precise role it plays in determining axonal polarity must be deﬁ ned, as well as the need to inhibit GSK-3 either in this entire compartment or just locally, as suggested from other studies [17].

3.4

HOW IS GSK-3 ACTIVITY CONTROLLED?

Although for many years GSK-3 was believed to be a constitutively active kinase, it has since become apparent that the activity of GSK-3 may be regulated by several different means. One regulatory mechanism that is still not fully understood appears to involve the interaction of the kinase with GSK-3 binding proteins [18] denominated FRATs. Three different FRATs have been cloned and characterized, and some contradictory data have been obtained regarding their inﬂuence on GSK-3. While in Xenopus, FRAT1 appears to act as an inhibitor of GSK-3 signaling, in some substrates FRAT 2 appears to preferentially increase GSK-3 mediated phophorylation of given residues. However, recently it was shown that FRATs are dispensable for Wnt signaling because the triple FRAT-knockout mouse does not display any major defects in brain development [19]. All these data indicate that there is still some work to be done before the precise role of FRATs in regulating GSK-3 can be established. However, the data available does suggest that the role of FRAT proteins in regulating GSK-3 neuronal function may not be so critical, or that it is compensated by other regulatory systems. Alternatively,

PATHWAYS CONTROLLING GSK-3 ACTIVITY IN NEURONS

51

these proteins may only become relevant under speciﬁc circumstances such as stress. An alternative regulatory mechanism is thought to involve the phosphorylation of speciﬁc GSK-3 residues by other kinases or through autophopshoryation [20]. Two different residues undergo phosphorylation in GSK-3, a serine (21α and 9β) and a tyrosine residue (279α. and 216β) that are present in both isoforms at the positions indicated. Indeed, it has been demonstrated that phosphorylation of serine 9/21 in GSK-3 is correlated with the inhibition of its kinase activity [21]. In contrast, tyrosine phosphorylation appears to be associated with an increase of its kinase activity [22]. Many kinases are involved in the serine phosphorylation of GSK-3, including Akt, ILK, PKA, and p90Rsk. Furthermore the inhibition of GSK-3 has been correlated with serine phosphorylation in many physiological situations, such as stimulation of the Insulin/IGF1, NGF, and Estradiol pathways [23]. However, there are fewer candidates that might be responsible for tyrosine phosphorylation of GSK-3. In neurons, the Pyk-2 and fyn kinases are capable of phosphorylating GSK-3 on tyrosine in vitro [24,25], and more recently MEK1/2 has been shown to fulﬁ ll this role in ﬁbroblasts. Furthermore in Dictyostelium there is compelling evidence that ZAK1 is responsible for the tyrosine phosphorylation of GSK-3 [26]. In neurons, tyrosine phosphorylation of residue 216 or 279 appears to increase under certain pathological situations upon exposure to FGF [27] or LPA [22]. Interestingly this was also observed following the exposure of neurons to β Amyloid or PrP [28,29], suggesting that the increase in kinase activity may be correlated with neuronal degeneration in some circumstances. More recently an alternative hypothesis has been proposed for the regulation of GSK-3 tyrosine phosphorylation, in which the incorporation of phosphotyrosine into GSK-3 might reﬂect intramolecular autophosphorylation in mammalian systems [30]. Such a process could play an important role in stabilizing the kinase, although our data indicate that not all pharmacological inhibitors of GSK-3 diminish its tyrosine phosphorylation (Simon, D, et al., submitted). Thus, in addition to the other regulatory systems proposed, we believe that the tyrosine phosphorylation of GSK-3 may be more precisely controlled by tyrosine kinases and phosphatases in mammalian system.

3.5

PATHWAYS CONTROLLING GSK-3 ACTIVITY IN NEURONS

Since many different pathways have been described in which GSK-3 plays an important or essential role, we will only summarize some of these here. Historically GSK-3 fulﬁlls a signiﬁcant role in the Insulin/IGF1 and Wnt/Shaggy signaling pathways. However, more recently it has become clear that GSK-3 is present in many other pathways such as those involving NGF, Estradiol, or Reelin. Moreover it seems likely that there will be many more in which the

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ROLE OF GSK-3/SHAGGY IN NEURONAL CELL BIOLOGY

relative importance of GSK-3 remains to be discovered. Here we focus our attention on those that are particularly relevant in neurobiology. 3.5.1

Insulin/IGF1

The Insulin/IGF1 system has been widely studied in neuronal and nonneuronal cells [4] as it is a pathway that controls processes such as survival apoptosis. In this pathway the inhibition of at least one pool of GSK-3 is essential for the survival of certain types of neuron. Furthermore it has been established that the PKB/Akt kinase exerts a negative inﬂuence on GSK-3 activity in this pathway through serine phosphorylation [21]. The serine phosphorylation of GSK-3 by ILK has also been established as an alternative regulatory event in other circumstances. In addition to the role of this Ins-IGF1/PI3K/Akt pathway in survival, this pathway also appears to control another important event in neuronal physiology, that of axonal elongation [15]. Indeed, it has been demonstrated that PI3K controls a complex formed by Par3/Par6/aPKC, which is essential in the formation of the axon in hippocampal neurons. This pathway may control some important elements in this process such as the activation of the Cdc42/Rac pathway. Thus, we can hypothesize that GSK-3 may be regulated by this complex, although evidence for such link only exists in the control of astrocyte migration [31]. It has previously been shown that in granule neurons that have already extended an axon, the inhibition of PI3K induces the collapse of the growth cone and the retraction of the neurite. This retraction may be prevented, at least in part, by the pharmacological inhibition of GSK-3 [32]. These data suggest that the activity of the PI3K/Akt pathway is essential not only for the determination of axonal polarity but also for the maintenance of the elongated axon, at least in some neurons. Nevertheless, the relevance of this pathway may vary between neuronal types or different developmental periods. 3.5.2

Wnt Signaling

Wnt signaling has been widely studied and it has been shown to be essential in early embryonic patterning, cell fate, cellular polarity and cell movement in both vertebrates and invertebrates [33]. In many, if not all, cell systems the so-called canonical Wnt pathway is formed by a set of phylogenetically conserved proteins including the Wnt receptor Frizzled (fz), and Disheveled (Dsh), a scaffolding protein that activates a complex formed by Axin/APC/ GSK-3-β/β˜ catenin. In this pathway GSK-3β phosphorylates β˜ catenin, among other proteins, this phosphorylation constituting part of a degradation signal for β˜ catenin. When Wnt binds to fz, Dsh inhibits the activity of GSK-3β in a manner that is not fully understood. This system appears to be speciﬁc to GSK-3β as no counterpart has been described for GSK-3α to date.

PATHWAYS CONTROLLING GSK-3 ACTIVITY IN NEURONS

53

In the mouse, 19 Wnt and 9 different Fz proteins have been identiﬁed and many of these have been found in brain tissue or during brain development. Indeed, Wnt’s appear to be essential in the regionalization of the forebrain, in the dorso-ventral patterning of the forebrain, and in hippocampal development. Furthermore evidence from Wnt7a mutant mice and the effects of Wnt7a in cell culture strongly support the hypothesis that some Wnt’s may play an additional and important role in cerebellar synaptogenesis [34]. This particular system has been discussed extensively and in more detail in Chapter 2 of this volume. 3.5.3

Estradiol Signaling

In neurons, estrogen regulates many processes, some as important as differentiation or survival. Transcriptional activity mediated by estrogen receptors is one of the best-studied hormonal actions. More recently it has been shown that in addition to its direct transcriptional activity, estrogen receptors activate a set of cytoplasmic signals in a similar manner to growth factors. Hence it has been reported that estradiol acts synergistically with IGF 1 in the brain or in neurons, activating the PI3K/Akt pathway [35]. Indeed, recent studies have demonstrated that the alpha but not the beta estrogen receptor interacts with the IGF receptor and with the regulatory subunit of PI3K, p85 [36]. A similar complex of estrogen receptor, GSK-3 and beta-catenin, has been recently reported in the adult brain, as well as in hippocampal or cortical neurons [37]. More important, the addition of this hormone increases the serine phosphorylation of GSK-3. This inhibitory phosphorylation is time and concentration dependent, and an antagonist of estradiol prevents this event. The kinase responsible is sensitive to the inhibition of the PI3K pathway and for this reason it seems that the best candidate is Akt (O. Varea et al., unpublished data). More remarkably, this hormone-dependent GSK-3 inhibition correlates with the transient stabilization of beta-catenin, which is released from a complex with the estrogen receptor and GSK-3. These data suggest that estradiol, in addition to its receptor-mediated transcriptional activity, may also regulate some neuronal functions by controlling the “pool” of beta-catenin [37]. 3.5.4

NGF Signaling

Through their speciﬁc tyrosine kinase receptors (TRKs), neurotrophins strongly activate two important pathways in neurons, one involving Raf/Mek/ Erks and the other PI3K/Akt. One of the roles of the PI3K cascade is to control and regulate the dynamics of the actin network. Similar signaling cascades have been described for other neurotrophins, and in these cases we can postulate that part of the survival system triggered by neurotrophins may be based on the action of the PI3K/Akt kinases. These may indeed be

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responsible for activating pro-survival signals and maintaining some proapoptotic elements, such as GSK-3, partially inhibited [21]. More recently it has been reported that NGF regulates microtubules in the axon and speciﬁcally, some MAPs that are thought to mediate axonal elongation through the inactivation of GSK-3β, such as APC. This hypothesis is supported by the fact that GSK-3β inactivation is required for efﬁcient axonal elongation and that this inactivation is mediated by the ILK kinase [17]. 3.5.5 Reelin Pathway There is considerable data to support the role of Reelin in brain development, speciﬁcally in cortical organization [38]. Reelin appears to exert its function through a complex receptor that is not well characterized, but in which some elements such as the EpoE receptor and the very low density lipoprotein receptor (VLDLR) are thought to be involved. The binding of Reelin appears to instigate signals in which the phosphorylation of Dab1 and that activation of GSK-3 has been implicated [39]. In this pathway the function of GSK-3 appears to be associated with the role of CDK5; however, the speciﬁc system that controls GSK-3 activity has yet to be deﬁ ned [39]. 3.5.6 LPA as Regulator of Neuron Morphology through Inﬂuence on the Cytoskeleton Lysophosphatidic acid (LPA) is a bioactive lipid with that can act as a growth factor. LPA binds to and transmits signals through three speciﬁc seven transmembrane domain receptors initially denominated as endothelial differentiating gene (EDG) and more recently re-named as lpa 1(EDG2), lpa 2 (EDG-4), and lpa 3(EDG-7; reviewed in [40]). Each of these couples to a speciﬁc downstream G-protein mediated signaling pathway. In neurons, LPA has been shown to induce neurite retraction and the rounding up of neuroblastoma cell lines [41]. In some primary neurons it also promotes growth cone collapse and neurite retraction [42]. Most important, LPA has been shown to control the morphology of cortical neuroblasts in the ventricular zone (vz) during neurogenesis, and indeed, lpa1 was cloned and characterized from embryonic brain [43]. The signaling pathways controlled by these three different receptors are not completely understood, although it has been demonstrated that they selectively activate different sets of heterotrimeric G proteins: Gi, Gα12/13, and Gαq/11 [44]. LPA-induced neurite retraction is blocked by the C3 transferase toxin, which ADP-ribosylates and inhibits Rho, indicating that this is a process that is mediated by this GTPase. Accordingly, activated Gα12 and Gα13 induce cell rounding through RhoA activation in neuronal cells [22,45]. In PC-12 cells, the activation of RhoA by Gα12 and Gα13 is mediated by different signaling pathways, one involving a tyrosine kinase sensitive to Tyrphosptin A25 (Gα13) and the other that is independent of such kinases. Additionally

SIGNIFICANCE OF GSK-3 ACTIVATION

55

Y-27632, a Rho-kinase inhibitor, blocks LPA-induced neurite retraction. Together these data indicate that this process involves the activation of a Gα12 /Gα13 -Rho-A-Rho-kinase [46]. We have characterized the LPA-response in different neuronal cell systems, focusing our attention on elements that could contribute to changes in MT dynamics during neurite retraction. Thus we have analyzed events such as the posttranslational modiﬁcations of tubulin and the phosphorylation level of MAPs. Additionally we analyzed whether the phosphorylation levels of different MAPs were altered in neurons during LPA-induced neurite retraction. These studies were performed on axonal MAPs, MAP1B, and Tau, and as a result we found that GSK-3 may be responsible for some of the phosphorylation detected in the LPA-retraction processes [22,45]. The lpa1 (EDG-2) receptor is differentially coupled to three different G proteins: Gi, Gα12/13, and Gαq. Considering that the neuronal cells selected express EDG-2, GSK-3 activation by LPA could be controlled by any of these G proteins or alternatively, this activation of GSK-3 might be controlled by a different pathway. We have addressed this question through a pharmacological approach, whereby we analyzed whether the interference with different elements located in the putative LPA-triggered pathways blocked LPAinduced GSK-3 activation and Tau hyperphosphorylation. As a result we demonstrated that LPA-induced hyperphosphorylation of Tau and GSK-3 activation was only interfered with when elements thought to be under the control of Gα12/13 were blocked. These results suggest that GSK-3 activation may be downstream Gα12 or Gα13. This possibility was conﬁrmed by overexpressing a GTPase dominant-form of Gα12 and Gα13 and measuring GSK-3 activity in transfected cells. Both active Gα12 and Gα13 induced GSK-3 activation through different molecular mechanisms [45]. The mechanisms by which Gα12/13 activates GSK-3 remains unclear; however, it might involve a tyrosine kinase as the increase of GSK-3 activity was correlated with additional tyrosine phosphorylation. Additionally it has been suggested that the tyrosine kinases Fyn and Pyk2 may phosphorylate GSK-3 [24,25]. Further studies will be necessary to provide a more complete view of how G proteins may control GSK-3 activity.

3.6

SIGNIFICANCE OF GSK-3 ACTIVATION

It has generally believed that GSK-3 activity is only negatively regulated and that this occurs through the insulin and Wnt pathways. However, its has recently been shown that GSK-3 can also be activated in response to some extracellular stimuli [24,39,49]. Direct measurements of GSK-3 activity in control and LPA-treated neurons, conﬁ rmed that GSK-3 activity increases during LPA-induced neurite retraction [46]. Furthermore we more recently found that Reelin and Netrin augmented GSK-3 activity [39,47]. Thus GSK-3 activation by LPA, Netrin, or Reelin seems not to be a particular

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characteristic of the cell line used but rather a more general physiological process. Indeed, even in situations where the ﬁ nal balance is an inhibition of GSK-3 kinase activity, such as following the addition of IGF1/Insulin or after estradiol addition, a transient activation of GSK-3 could be observed [24,37]. All these data suggest that the upregulation and downregulation of this kinase is more complex than might initially have been considered. Indeed, in Dyctiostelium discoideum it has already been shown that GSK-3 activity may increase in response to cAMP binding to an heptahelical G-protein coupled receptor. In this system a tyrosine kinase and a tyrosine phosphatase have been described as regulators of GSK-3 activity [26,48]. 3.7 PUTATIVE PHYSIOPATHOLOGICAL IMPLICATIONS OF GSK-3 ACTIVATION In certain neurodegenerative processes, such as Alzheimer’s disease (AD), GSK-3 appears to be deregulated. Indeed, it is thought to be responsible for the aberrant phosphorylation that contributes to neurodegeneration and to augmenting the formation of neuroﬁbrillar tangles, a pathological hallmark of AD. Furthermore GSK-3 is one of the kinases activated by β˜ Amyloid [6,28] or PrP [49]. It is for this reason that GSK-3 is emerging as a promising therapeutic target in neurodegenerative processes such as AD and ischemia [50], as well as in psychiatric disorders. Thus understanding the regulatory mechanisms that control the activation and inhibition of GSK-3 is an increasingly more important issue. 3.8 CONCLUDING REMARKS GSK-3 was initially identiﬁed as one of the serine/threonine protein kinases that phosphorylates and inhibits glycogen synthase. It now still more evident that GSK-3 plays an important role in several physiological processes such as cell cycle, apoptosis, and development. In this respect numerous data indicated that GSK-3 plays a essential role in neuronal morphogenesis and neuronal survival. However, we still have many open questions, such as the possible function of each isoform, in these neuronal processes. Although for many years GSK-3 was believed to be a constitutively active kinase, it has since become apparent that the activity of GSK-3 may be regulated by several different pathways. As a general role many pathway control GSK-3 by inhibition, which in most cases correspond with a “boots of survival.” However, the transient activation, or a more persistent activation, appears not necessarily correlated with neuronal death. Even if we consider that data from many labs indicated that deregulation of GSK-3 it is associated with some pathological processes such as Alzheimer’s disease (AD) or ischemia, the regulatory possibilities have to be fully clariﬁed. While we have a “good picture” of this intriguing kinase, we feel that more is still to come.

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4 THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3 Ernst ter Haar Vertex Pharmaceuticals Incorporated, Cambridge, MA

Contents 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

4.1

Crystal Structures of GSK-3β Phosphorylation Sites of GSK-3β The Primed-Phosphorylation Mechanism Auto-inhibition by GSK-3 N-Terminus ATP Binding Site Crystal Structures of GSK-3β Inhibitor Complexes Structures with Wnt Signaling Pathway Peptides Concluding Remarks

CRYSTAL STRUCTURES OF GSK-3b

Mammalian cells contain two major isoforms of GSK-3, GSK-3α, and GSK-3β that have virtually identical kinase domain sequences; however, the protein sequences diverge at the N- and C-terminii [1–3]. The sequences of the kinase domains are 90% identical and 97% homologous. At the N-terminus GSK-3α has a 63-residue glycine-rich insertion (71% glycine). At the C-terminus the sequence homology of the last 80 residues drops to 34%. There is one additional GSK-3β isoform that contains a 13 amino acid insertion in the kinase Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

61

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THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

domain [4]. This isoform is found mostly in the brain, and its role has yet to be determined. Three-dimensional atomic structures of proteins have proved to be very helpful in elucidating protein function. Comparisons of the structure with related proteins reveal similarities and differences that explain the intricate workings of the molecules. Atomic structures are also very helpful in explaining the role of physiologically relevant mutations, identifying new binding sites for protein interactions and explaining the structure activity relationships of small molecule inhibitors bound at the ATP binding site. The ﬁ rst crystal structures of GSK-3β were published in 2001 [5,6]. These structures revealed that this protein has a typical serine/threonine kinase fold with a small N-terminal domain (residues 25 to 134) and a larger C-terminal domain (residues 135 to 380) (Fig. 4.1). The N-terminal domain consists of seven antiparallel β-strands and the α-C helix. β-Strands two through six form an antiparallel β-barrel that is interrupted by the α-C helix (residues Arg96 to Met101) between β-strands ﬁve and six. The ATP binding site is located at the interface of the N-terminal and C-terminal domains and is enclosed by the glycine-rich loop (residues 60 to 70) and the hinge (residues 134 to 139). The activation loop starts with the DFG motif (Asp200 to Gly202), ends with the APE motif (Ala224 to Glu226) and forms one edge of the substrate-binding groove [7]. The other edge is formed by the loop that connects β-strand ﬁve with the α-C helix. The ﬁ nal 55 residues after the kinase domain (residues 330 to 384) form a cluster of loops and helices that packs against the C-terminal domain. The Protein Data Bank contains the coordinates of 14 GSK-3β structures; two each of apo-GSK-3β and GSK-3βpeptide complexes and 10 GSK-3β complexes with small molecules in the ATP binding site (Table 4.1). 4.2

PHOSPHORYLATION SITES OF GSK-3b

The kinase fold of GSK-3β superimposes well on the structures of MAP kinases p38 and ERK2 and also CDK2. The catalytic activity of these kinases depends on the correct alignment of the N- and C-terminal domains, which is usually modulated by two phosphorylated residues in the activation loop. One of the phosphorylated residues (typically a phospho-threonine) binds positively charged residues from the N-terminal and C-terminal domains via its phosphate group. Phosphorylation of the second residue (typically a phospho-tyrosine) opens up the substrate-binding groove. GSK-3β has two phosphorylation sites that regulate the catalytic activity, Ser9 and Tyr216. AKT phosphorylates GSK-3β at Ser9 attenuating enzyme activity. This mechanism of inactivation by phosphorylation is speciﬁc for the insulin signaling pathway and is not used in the Wnt signaling pathway [8,9]. GSK-3β has only one phosphorylation site in the activation loop, Tyr216, as opposed to two in MAP kinases. A valine (Val214) occupies the place of the phospho-

63

PHOSPHORYLATION SITES OF GSK-3β

1 2

S25 2

GLYCINE-RICH LOOP ATP BINDING SITE

3

3 5

4

4

ATP BINDING SITE

α-C HELIX

Y216

GLYCINE-RICH LOOP

R383 ACTIVATION LOOP

HELICAL CLUSTER (RESIDUES 330-383)

A

B

Figure 4.1 Crystal structure of GSK-3β GSK-3β is a 420-residue serine/threonine kinase. (A) The crystal structure (pdb : 1i09) encompasses residues 25 to 383 and reveals the characteristic kinase features, such as the glycine-rich loop, the α-C helix, the activation loop and the ATP binding site. GSK-3β has two phosphorylation sites: Ser9 and Tyr216. Ser9 is an auto-inhibition site that inhibits the catalytic activity when it is phosphorylated. This phosphorylation site is disordered in GSK-3β crystals and could not be identiﬁed in the crystal structure. Tyr216 is the phosphorylation site in the activation loop. Phosphorylation of Syr216 increases the catalytic activity of GSK3β. (B) The structure was rotated 90˚ along the vertical axis.

TABLE 4.1 PDB Code 1h8f 1i09 1gng 1o9u 1pyx 1j1b 1j1c 1q3d 1q3w 1q41 1q4l 1q5k 1r0e 1uv5

Description of Structure

Reference

apo GSK3β apo GSK3β phosphorylated (Y216) GSK3β with Frat1 peptide phosphorylated (Y216) GSK3β with Axin peptide GSK3β with AMPPNP GSK3β with AMPPNP GSK3β with ADP GSK3β with Staurosporine GSK3β with Alsterpaullone GSK3β with Indirubin-3’-monoxime GSK3β with I-5 (3-anilino-4-arylmaleimide) GSK3β with AR-A014418 GSK3β with 3-indolyl-4-aryl maleimide GSK3β with 6-Bromoindirubin-3’-oxime

[6] [5] [11] [10] [31] [32] [32] [31] [31] [31] [31] [45] [44]

threonine. The Tyr216 phosphorylation site is conserved in many kinases and is located in the activation loop. Both these phosphorylation sites are also present in GSK-3α where Ser21 is the inactivating phosphorylation site and Tyr279 is the activation loop phosphorylation site. The crystal structure of Tyr216 phosphorylated GSK-3β conﬁ rms the role of pTyr216 as a gatekeeper

64

THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

of the substrate-binding groove (PDB 1GNG and 1O9U) [10,11]. Compared with the apo GSK-3β structure, the phospho-phenol side chain of pTyr216 has rotated out of the substrate-binding groove and is held in place by two arginine side chains (Arg220 and Arg223) (Fig. 4.2). Phosphorylation of Tyr216 increases the catalytic activity 5-fold and, in agreement, the Y216F mutant exhibits 5- to 10-fold lower activity. These modiﬁcations affect the rate of the reaction (kcat) but do not affect the afﬁ nity of the enzyme for the substrate (K M) [10]. Bhat et al. [12] transfected the catalytically inactive mutant GSK-3β–K85R into PC12 cells to study the Tyr216 phosphorylation. GSK-3β becomes activated via Tyr216 phosphorylation when the cells undergo apoptosis. These apoptotic responses were induced through NGF withdrawal or staurosporine exposure. The kinase-inactive mutant GSK-3β–K85R was still phosphorylated on Tyr216 in response to the apoptotic signals, which suggests the involvement of an upstream kinase. Tyrosine kinase PYK2 was identiﬁed as phosphorylating Tyr216 by overexpressing the kinase with GSK-3 in CHO cells [13]. Wild-type PYK2, and not the kinase dead mutant, phosphorylated GSK-3β on Tyr216. PYK2 coimmunoprecipitated with GSK-3 and PYK2 phosphorylated GSK-3 in vitro. FYN also immunoprecipitated with GSK-3β in response to insulin treatment of SH-SY5Y cells, and puriﬁed FYN phosphorylated Tyr216 of GSK-3β [14]. Cole et al. postulated the autophosphorylation theory [15] by showing that Tyr216 is not phosphorylated when catalytically inactive forms of GSK-3β are expressed in HEK-293 cells or E. coli. Furthermore the GSK-3 isoforms become dephosphorylated when the cells are exposed to Kenpaullone or Indirubin, two GSK-3β speciﬁc inhibitors that bind in the ATP site. They also provide evidence of tyrosine autophosphorylation in vitro. The extent of Tyr216 phosphorylation was independent of the protein concentration, and wild-type GSK-3β could not tyrosine phosphorylate kinase-dead GSK-3 mutants, suggesting that autophosphorylation was via an intramolecular process. From the structure of apo-GSKβ, it is not obvious how an intramolecular phosphorylation could take place at Tyr216. A recent study of activation loop autophosphorylation in DYRK suggested that transitional intermediates of the nascent kinase are capable of intramolecular tyrosine phosphorylation and that the tyrosine kinase phosphorylation capacity is lost in mature DYRK [16]. A similar mechanism of action would explain why the autophosphorylation activity in GSK-3β is seen in cells expressing GSK-3β, but not in in vitro phosphorylation assays.

4.3

THE PRIMED-PHOSPHORYLATION MECHANISM

Though it bears the name for its phosphorylation activity on glycogen synthase, GSK-3 is involved in many different signaling pathways and has numerous different substrates. One reference counted more than 40 GSK-3 substrates

THE PRIMED-PHOSPHORYLATION MECHANISM

65

A

B

C

Figure 4.2 Activation loop phosphorylation site. Tyr216 is the phosphorylation site located in the activation loop. (A) When Tyr216 is not phosphorylated, its side chain occupies the substrate-binding groove. (B) The side chain of Tyr216 rotates out of the substrate-binding groove when Tyr216 is phosphorylated. The phosphate group is held in place by two arginine side chains: Arg220 and Arg223. (C) The tyrosine phosphorylation site is also found in other map kinases, such as PTtyr185 in Erk2 (pdb : 2erk). This phosphorylation site is often accompanied by a threonine phosphorylation site, which is absent in GSK-3β.

66

THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

and the list is still growing [17]. GSK-3 does not phosphorylate all the substrates in the same manner or with the same efﬁciency. GSK-3 phosphorylates many of its substrates via a primed-phosphorylation mechanism, recognizing the canonical phosphorylation motif SXXXpS. This motif contains the phospho-accepting Ser or Thr that is separated by three residues from a phospho-serine or phospho-threonine. The phosphorylation mechanism is called primed-phosphorylation because a different kinase must ﬁ rst phosphorylate the substrate at the P+4 position, before GSK-3 can phosphorylate the P0 residue. The priming kinase varies for each substrate. For instance, CDK5 is the priming kinase for Tau and phosphorylates Ser235 [18,19], while GSK-3β subsequently phosphorylates Thr231. Although GSK-3β also phosphorylates Tau at a site that does not require priming [20,21] phosphorylation at the primed site reduces the interaction between Tau and microtubules [22]. The priming kinase for glycogen synthase is casein kinase II. Glycogen synthase has the primed phosphorylation motif repeated four times (residues 640, 644, 648, 652, and 656) and GSK-3β will phosphorylate all four Ser residues sequentially after casein kinase II has phosphorylated Ser656 [23,24]. The primed phosphorylation of the GSK-3 substrate is necessary to align the two kinase domains. Other kinases such as ERK2 [25], p38.γ [26], and CDK2 [27] have a phospho-threonine for this purpose, but GSK-3 lacks that phosphorylation site in the activation loop (Fig. 4.2). In the case of GSK-3, the phospho-group of the substrate makes contact with the positively charged residues Arg96, Arg180, and Lys205 just as the phospho-threonine in MAP kinases interacts with the positively charged residues. This similarity was discovered in the ﬁ rst two reported crystal structures of GSK-3β based on some unusual observations. One of the reported structures had a phosphate ion (PDB 1I09) [5], and the other had a HEPES molecule binding the positively charged residues (PDB 1H8F) [6]. It was immediately recognized that these negatively charged moieties occupy the site that the phosphate in the primed peptide could ﬁ ll. In subsequent structures of GSK-3–ligand complexes, a sulphate (PDB 1GNG) [10] and a citrate ion (PDB 1R0E) have also been observed occupying the same site. Because the substrate of GSK-3β provides the phosphorylated residue to align the two domains, the substratebinding groove of GSK-3β is extended and traverses beyond the loop that contains residues Lys91 to Lys93. MAP kinases that have the threonine phosphorylation site in the activation loop have a shorter substrate-binding groove (Fig. 4.3). The structure of phosphorylated and peptide-bound CDK2 (PDB 1QMZ) shows that the substrate-binding groove ends near the tip of the glycine-rich loop.

4.4

AUTO-INHIBITION BY GSK-3 N-TERMINUS

AKT phosphorylates Ser9 leads to inactivation of GSK-3β [28,29]. In GSK3α, Ser21 is the homologous site but is separated from the kinase domain by

AUTO-INHIBITION BY GSK-3 N-TERMINUS

67

Figure 4.3 Comparison of the substrate-binding grooves of GSK-3β and Cdk2. The structures of phosphorylated GSK-3β (pdb : 1gng) and Cdk2 in complex with a peptide (pdb : 1qmz) were used to show the differences between the substrate-binding grooves of GSK-3β and other map kinases. GSK-3β was super imposed onto Cdk2 to model the peptide in the GSK-3β substrate-binding groove. The substrates of GSK-3β provide the phosphate group that is used by serine/threonine kinases to align the Nterminal and C-terminal domains. A consequence of this phosphorylation mechanism is an expanded substrate-binding groove.

a 63-residue long glycine-rich insertion. The ﬁ rst 25 residues of the GSK-3β crystal structure were disordered, and hence the position of Ser9 cannot be determined. Binding studies with a peptide based on the GSK-3β aminoterminus (residues 3 to 12) that contained the phosphorylated Ser9 established that auto-inhibition of GSK-3β is due to binding of the phosphorylated Ser9 peptide in the substrate-binding groove [6]. This interaction was not only dependent on the serine phospho-group but also on the surrounding sequence as an unrelated phospho-serine peptide did not inhibit GSK-3β activity. Despite this sequence requirement the inhibitory potency of the peptide is weak (Ki of 700 μM). Since the peptide is part of GSK-3β, the effective concentration would be much higher to overcome the weak afﬁ nity [6]. On the other hand, binding of the auto-inhibitory motif should be readily reversible to serve as substrate for phosphatases and regain its activity. According to the model presented by Dajani et al. and Frame et al. [6,30], the N-terminus of GSK-3β binds in the substrate-binding groove as if it contains the canonical phosphorylation motif SXXXpS. Phosphorylated Ser9 binds at P+4 site and makes contact with the positively charged residues. The phospho-accepting site or P0 site is occupied by Pro5. A proline, like serine or threonine, has a small side chain and can easily be accommodated at that position [6]. Further proof that Pro5 occupies the phospho-accepting site comes from the ﬁ nding that an 11-mer peptide derived from the GSK-3β N-terminus (residues 4–14) is phosphorylated when Pro5 is changed to a serine [30]. Residues 10 to 24 form the linker between Ser9 and the N-terminal domain. Fourteen residues are enough to bridge the distance between the P+4 site and the ﬁ rst ordered residue in the GSK-3β crystal structure, which is residue 25.

68

4.5

THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

ATP BINDING SITE

The ATP binding site is a well deﬁned and fairly deep pocket at the interface of the N-terminal and C-terminal domains (Fig. 1A). This binding site is conserved among protein kinases. The Protein Data Bank has two structures of GSK-3β in complex with the nonhydrolyzable ATP analogue AMPPNP (PDB 1PYX and 1J1B) [31,32]. The structures show how the catalytic residues of the γ-phosphate transfer area form a large network of interactions with the AMPPNP phosphates (Fig. 4.4A). The binding pocket can be visualized as ﬁve distinct regions making speciﬁc interactions with the ATP-Mg 2+ complex: the phosphate transfer area, the glycine-rich loop, the upper hinge region, the lower hinge region and the hydrophobic pocket (Fig. 4.4B). Lys85 is positioned between the α- and β-phosphates of the ATP analogue and forms a salt bridge with Glu97 (Fig. 4.4A). It also interacts with the Asp200 side chain, which orients one of the Mg 2+ ions that is located between the β- and γ-phosphate groups and two water molecules. Asn186 orients the second Mg 2+ ion, which makes contact with the α- and γ-phosphates and the carboxylate group of Asp200. The Asn186 side chain amide makes a hydrogen bond with the backbone carbonyl of Asp181. The carboxyl side chain of

Figure 4.4 (A) A large network of hydrogen bonds connects ATP with the phosphorylation substrate and facilitates the γ-phosphate transfer. The glycine-rich loop (residues 60 to 70) has been removed from the structure for clarity. (B) A surface rendering of the GSK-3β ATP binding site. A kinase ATP binding site can be divided into different regions that each have their own characteristics. The phosphate-transfer region is highly conserved in kinases. The hydrophobic pocket has a lot of sequence variation that is often targeted to increase the selectivity of inhibitors in kinase drug design programs. The glycine-rich loop is a ﬂexible lid that covers the ATP binding site. The upper hinge makes hydrogen bonds with ATP or other small-molecule ligands that occupy the ATP binding site. The lower hinge forms the edge of the solvent opening.

ATP BINDING SITE

69

Asp181 abstracts the proton from the hydroxyl of the substrate serine or threonine and orients the Lys183 side chain. The amino group of Lys183 is believed to assist in the transfer the γ-phosphate from ATP to the accepting serine or threonine side chain. The network of interactions with the nucleotide is also partly responsible for aligning the N-terminal and C-terminal domains. While Glu97 forms a salt bridge with Lys85, its neighbor, Arg96 binds the phosphate group of the primed serine in the substrate-binding groove. Asp181 orients the side chain of Lys183, while Arg180 also binds the phosphorylated serine in the substrate-binding groove. Therefore the alignment of the kinase domain is closely coordinated with the γ-phosphate transfer. The glycine-rich loop is the ﬂexible lid of the ATP binding site. It is composed of β-strands three and four of the anti-parallel β-barrel of the Nterminal domain. The ﬂexibility comes from the presence of three conserved glycines that permit the loop to adjust and conform to the size of the molecule in the ATP binding site. The adenine moiety of AMPPNP makes Van der Waals contacts with the two hydrophobic residues of the glycine-rich loop, Ile62 and Val70, and is wedged between Ala83 and Leu188. At the tip of the glycine-rich loop is Phe67. This bulky hydrophobic residue can be solvent exposed, but it can also be buried together with the inhibitor by the glycinerich loop, depending on the molecule in the ATP binding site. Phe67 is completely buried by the glycine-rich loop in the GSK-3β-3-indolyl-4-aryl maleimide complex (PDB: 1R0E). In the GSK-3β-AMPPNP complex the phenyl group of Phe67 is solvent exposed because the phosphates of AMPPNP are buried by the glycine-rich loop and create an unfavorable environment for the phenyl ring. The hinge region (residues 133 to 139) is the ﬂexible linker between the N-terminal and C-terminal domains and allows the two domains to rotate with respect to each other (Fig. 4.4A). The residues of this region make hydrogen bonds with the adenine moiety of ATP via their backbone atoms, a pattern typical for kinase ATP binding sites. At the start of the hinge, a backbone carbonyl (Asp133 in GSK-3β) acts as an H bond acceptor and forms a hydrogen bond with the N6 amine of ATP. The next residue is not involved in H bonds. But the subsequent residue (Val135 in GSK-3β), makes two hydrogen bonds with the adenine moiety. The backbone amide of Val135 donates its hydrogen to N1 of ATP. The backbone carbonyl also makes an hydrogen bond with the adenine moiety. This is not always recognized as such, but aromatic carbons in heterocyclic rings can be the donating contributor in an hydrogen bond [33]. The hydroxyl group of Thr138 is indirectly linked via a bridging water molecule to the ribose hydroxyl of ATP. This bridging water molecule was also observed in co-complexes with small molecule inhibitors. In CDK2 (PDB 1QMZ), the side chain of Asp86 makes direct contact with the ribose group [27]. The backbone carbonyl of GSK-3β residue Gln185 makes an hydrogen bond with the O3′ ribose hydroxyl.

70

THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

4.6 CRYSTAL STRUCTURES OF GSK-3b INHIBITOR COMPLEXES GSK-3 has been considered a target for adult onset diabetes [34–36], stroke [37,38], Alzheimer’s disease [39,40], bipolar disorder [41], and schizophrenia [42,43]. The ATP binding site has been the preferred site for kinase drug design and the crystallization of inhibitors with GSK-3β is relatively straightforward. Unphosphorylated protein and ligand readily form diffracting crystals when combined with a mixture of PEG and salt (e.g., [5]). The PEGION screens from Hampton Research or Nextal Biotechnologies yield crystals under multiple conditions. The crystals typically have the same space group as the native unliganded protein, although exceptions have been observed (e.g., the GSK-3β complex with 6-bromoidurubin, PDB 1UV5 [44]). The space group is P212121 with unit cell dimensions 83, 86, and 127 Å, and 90˚ angles, and there are two GSK-3β inhibitor complexes in the asymmetric unit. The Protein Data Bank contains seven crystal structures of GSK-3β in complex with non-ATP inhibitors, all of which bind GSK-3β at the ATP binding site [31,44,45]. Although the ATP competitive inhibitors occupy the general area of the ATP binding site, they do explore other available space nearby depending on their structure. All ATP binding site inhibitors make hydrogen bonds with backbone atoms of the kinase domain hinge (residues Asp133 to Thr138) (Fig. 4.5). The hydrogen bonds are the same as observed with ATP, but different inhibitors make different combinations of hydrogen bonds. For instance, the two indirubin complexes (PDB 1UV5 and 1Q41) have four hydrogen bonds (Fig. 4.5). In contrast, the Alsterpaullone complex (PDB 1Q3W) only has three hydrogen bonds (with the two backbone atoms of Val135). Some GSK-3 inhibitors also target other areas of the ATP pocket. For instance, the nitro-group of the Alsterpaullone (PDB 1Q3W) and the chlorine of I-5 (3-anilino-4-arylmaleimide) interact with the conserved catalytic lysine, Lys85. The bromine atom of 6-bromoindirubin (PDB 1UV5) is buried in the hydrophobic pocket of GSK-3β among residues Leu132, Leu130, and Met101 [44]. This is a pocket that is often targeted to increase the selectivity of the inhibitor, since it is one of the most diverse areas in the ATP binding site of kinases and was successfully used to increase the selectivity in favor of p38α over ERK2 [46]. The GSK-3β ATP binding site inhibitors do not cover the γ-phosphate transfer area. Targeting this part of the ATP binding site does not appear to improve the selectivity of the inhibitor, although it may improve the potency as additional contacts between the inhibitor and the protein are established. Although the ATP binding site is well conserved in kinases, it is possible to obtain selective kinase inhibitors by taking advantage of the small differences that exist between the ATP binding sites. The indirubins not only inhibit GSK-3β but also cyclin-dependent kinases (CDK’s), a group of kinases that is structurally very similar to GSK-3β. Adding a sulphonic acid group to

71

CRYSTAL STRUCTURES OF GSK-3β INHIBITOR COMPLEXES

NH2 O HN

N

N

N

N

N

NH O O

HO

O H − O P O − P N O P O O O

HO

O

AMPPNP

N+

O−

Br O

O NH O N HN

HN

OH

HN

Alsterpaullone

O O−

O

HN

OH

6-Bromoindirubin-3′-oxime

Cl

O

O

O

O

HN

HN OH

HN

NH O

S

N

N

O

O

Indirubin-3′-monoxime

O

NH O NH

HN

O

NH

N H

HN

NH O

O

Staurosporine

O O

O

N

− OO

NH

OH

O

HN

F

O O

O

HN

N

Cl HO

O

O

AR-A014418

3-anilino-4-arylmaleimide (l-5)

3-indolyl-4-aryl maleimide

Figure 4.5 Structures of GSK-3β ATP binding site inhibitors. The protein data bank contains 10 crystal structures of GSK-3β inhibitor complexes. Seven of the 10 structures are non–ATP inhibitors belonging to different chemical classes. All the inhibitors make hydrogen bonds with the backbone atoms of the hinge residues.

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THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

the 5-position of indirubin increases the CDK2/cyclin A inhibition potency 63-fold; however, the GSK-3β inhibition potency only increases 2-fold. The crystal structure of CDK2 in complex with indirubin-5-sulphonic acid shows that the sulphonic acid is buried in a pocket that includes Ala144. The similar pocket in GSK-3β is less suitable for the sulphonic acid because the homologous residue is the bulkier Cys199, which creates a smaller pocket [31]. The lower portion of the hinge, together with the glycine-rich loop forms the solvent exposed region of the ATP binding site. The size of the solvent opening can vary because of interdomain movement. GSK-3 has two residues that effectively decrease the size of the solvent opening: Glu137 forms a salt bridge with Arg141. In kinase drug design programs the solvent opening has been successfully used to improve the pharmacological properties of inhibitors. Soluble groups such as morpholino and piperazine moieties can be tethered to the inhibitor at a position where it can protrude through the solvent opening (FGF receptor in complex with PD 173074) [47]. These moieties typically have little effect on the binding properties of the inhibitor. The Glu137–Arg141 salt bridge restricts the choice of locations where the tethered soluble groups can be added in GSK-3β inhibitors. A large group of kinases, especially tyrosine kinases have an additional glycine inserted in the lower portion of the hinge. Although this glycine insertion does not play a role in the catalysis of ATP, it has an effect on the shape of the ATP binding site solvent opening and the relative position of the upper hinge. When IGF1R (with the glycine insertion) [48] was superimposed onto GSK-3 (without the glycine insertion) by matching the catalytic loops (GSK3β residues 183 to 187 and IGF1R residues 1134 to 1138) and the DFG motifs (GSK-3β residues 200 to 202 and IGF1R residues 1150 to 1152), the upper hinge of IGF1R clearly had shifted 2.3 Å because of the glycine insertion (Fig. 4.6). The superposition of the GSK-3β inhibitor complexes onto the unliganded structure reveals the ﬂexibility of the GSK-3β kinase domain. Comparison of the GSK-3β–AMPPNP and GSK-3β–staurosporine complexes showed that the N-terminal domain of the staurosporine complex had rotated back by 10˚ [31]. The glycine-rich loop is a ﬂexible anti-parallel β-strand that adjusts its position depending on the ATP binding site occupant. Because of the Van der Waals interactions, the glycine-rich loop moves toward the C-terminal domain to interact with hydrophobic ligand moieties. When the ATP binding site is empty, the glycine-rich loop normally keeps the solvent opening more exposed and allows solvent molecules to occupy the binding pocket. Phenylalanine at the tip of the glycine-rich loop (Phe67) has been observed in multiple conformations. A second ﬂexible region is the loop between β-strand ﬁve and the α-C helix that forms the roof of the substrate-binding groove. It is not hard to imagine why this part is ﬂexible, since GSK-3β has many substrates with divergent amino acid sequences and most of them bind in the substrate-binding groove before undergoing phosphorylation. Such ﬂexibility aids in the docking of differently shaped substrates to GSK-3β. The β5-αC

STRUCTURES WITH WNT SIGNALING PATHWAY PEPTIDES

73

Figure 4.6 differences in hinge position due to glycine insertion. The structure of GSK-3β with amppnp (pdb : 1pyx) was superimposed onto tyrosine kinase IGF1R (pdb : 1ir3) by aligning the catalytic loops (GSK-3β residues 183 to 187 and IGF1R residues 1134 to 1138) and the DFG motifs (GSK-3β residues 200 to 202 and IGF1R residues 1150 to 1152). The glycine insertion of IGF1R displaces the hinge by 2.3 Å compared to the GSK-3β hinge.

loop is in close proximity to the area where different anions (phosphate, citrate, and a HEPES molecule) bind the substrate-binding groove. These anions occupy the position of the phosphate group of the primed residue. The anion-binding site is about 10 Å from the closest loop residue side chain (Phe93). The size of a phospho-serine residue is only 5.5 Å. The movement of the loop toward the phosphorylated peptide substrate helps maintain the same relative position for the phosphate.

4.7 STRUCTURES WITH WNT SIGNALING PATHWAY PEPTIDES The Wnt signaling pathway plays a key role in many developmental processes and has different effects in different organisms. In Drosophila, Wnt signaling affects segment polarity [49]. It affects axis induction in Xenopus and plays a role in carcinogenesis in humans (reviewed in [50]). The major components of the Wnt signaling pathway are APC (adenomatous polyposis coli), Axin,

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THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

β-catenin, GSK-3β, Wnt, Frizzled, and Disheveled [51]. The Wnt signaling molecule binds to the extracellular domain of the Frizzled receptor and activates the signaling pathway. This leads to increased β-catenin levels and subsequent transcription activation. In the absence of Wnt, the transcriptional co-activator β-catenin is phosphorylated by GSK-3β, then ubiquinated, and degraded by the proteosome. Axin is a large docking protein that binds APC, GSK-3β, casein kinase Iα, and the β-catenin [52–58]. Axin is essential for bringing all the components together to facilitate the β-catenin phosphorylation and degradation [56]. Casein kinase Iα is the priming kinase of β-catenin and phosphorylates Ser45. GSK-3β phosphorylates Thr41, Ser37, Ser33, and Ser29 [52] on β-catenin, but GSK-3β cannot phosphorylate β-catenin without Axin. When the Wnt signaling pathway is activated, the Axin–GSK-3β–βcatenin complex disintegrates and β-catenin escapes phosphorylation and degradation and accumulates in the cytoplasm and nucleus. Initially it was thought that β-catenin did not require primed phosphorylation, but that the scaffolding of β-catenin and GSK-3β was enough to ensure efﬁcient phosphorylation of β-catenin [59,60]. However, it turns out that βcatenin is also subjected to primed phosphorylation by casein kinase Iα [52]. Primed phosphorylation enhances the phosphorylation rate 30-fold, while scaffolding enhances the phosphorylation 24,000-fold. With these two mechanisms combined, the phosphorylation reaction is an astonishing 720,000 times faster than in the absence of Axin. The binding site of Axin on GSK-3β has been determined by X-ray crystallography using a peptide segment of Axin [10,11] (Fig. 4.7). Although Axin is a substrate of GSK-3β, the Axin peptide (residues 381 to 401) does not bind in the typical kinase substrate-binding groove, but in a groove between the α-G helix and the loop containing residues 286 to 296 of the C-terminal domain. The loop connects the α-H helix with the α-J helix and is often disordered in GSK-3β crystal structures. The disorder is due to multiple conformations of the loop in the GSK-3β molecules that make up the crystals. The binding of the Axin peptide (residues 383 to 400 in human Axin) to GSK-3β causes the loop to shift and open up the binding groove. The peptide binds as a single amphipathic α-helix, and the main interactions between the Axin peptide and GSK-3β are between hydrophobic residues. Phe388, Ala389, Leu392, and Leu396 are deeply buried in the binding groove and make hydrophobic contacts with Val263, Leu266, Val267, and Ile270 on one side and Tyr288, Phe291, Phe293, Pro294, and Ile296 on the other side. There are two additional polar contacts: Gln400 of Axin makes a hydrogen bond with the backbone carbonyl of Gln295, and the side chains of Arg395 (Axin) and Asp264 (GSK-3β) form a salt bridge [10]. Different groups have used truncated Axin constructs to ﬁnd the minimal portion that binds GSK-3β. Unfortunately, these experiments used Axin from different species that do not have identical sequences. Mouse Axin is 130 residues longer than human Axin and rat Axin is six residues longer. The GSK-3β

75

STRUCTURES WITH WNT SIGNALING PATHWAY PEPTIDES A

B

ATP-BINDING SITE SUBSTRATE BINDING GROOVE

AXIN FRATTIDE

D

C

pY216

pY216

R201 Q204

S261

I228 S261 E384 F388

D264

N287 VAL 383

L206

V267

E391

L208

L202

D264

V263

T289

E215 R219

R395 Q386 V399

I393 E397

E290

R218 K214

E390

F291

F293

K292 P294

K292

Figure 4.7 Axin and Frat1 binding sites. (A) The structure of phosphorylated GSK3β in complex with an Axin peptide (pdb : 1o9u). (B) The structure of phosphorylated GSK-3β in complex with Frattide (pdb : 1gng). The Axin (C) and Frattide peptides (D) bind the C-terminal domain of GSK-3β with mainly hydrophobic and some hydrophilic interactions. The peptide residues are indicated in black; the GSK-3β residues in black and white fonts.

phosphorylation sites of Axin are Thr481 and Ser486 in human Axin but Thr609 and Ser614 in mouse Axin. The picture that emerges is that Axin has different domains that bind GSK-3β, β-catenin, APC, or phosphatase PP2A and that the phosphorylation state of Axin is also a factor [56–58]. The phosphorylation site of Axin (Ser486) is located in the β-catenin binding domain. Axin needs to be phosphorylated at this site in order to bind β-catenin [61,62]. It is not clear whether GSK-3β needs to be catalytically competent to bind

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THE CRYSTAL STRUCTURES OF GLYCOGEN SYNTHASE KINASE 3

Axin. One study reported that the GSK-3β mutants K85M and Y216F could not bind Axin as determined by coimmunoprecipitation using HA-tagged GSK-3β and Myc-tagged rat Axin in COS cells [53]. A different study using microinjected kinase-dead Xenopus GSK-3β together with Myc-tagged Axin reported that the two proteins coimmunoprecipitated equally well [63,64]. Others have reported more subtle differences: Ser9 phosphorylated GSK-3β and the Y216F GSK-3β mutant bind Axin less efﬁciently [65]. Until recently it was thought that Frat1 played an important role in the Wnt signaling pathway. Studies have shown that Frat1 (or GSK-3-binding protein (GBP)) breaks up the GSK-3β, APC, β-catenin, Axin complex when the Wnt signaling pathway is activated [66]. The Frat1/Frat2/Frat3 triple knockout mice, however, turned out to be viable, healthy and without any overt phenotypic aberrations [67,68], suggesting that Frat1 is not a key regulator that prevents GSK-3β from phosphorylating β-catenin. The crystal structure of GSK-3β in complex with the Frat1 peptide (also named Frattide) was determined before the publication of the triple-knockout study [11]. Frat1 is 274 residues long. The 39-residue peptide is derived from Frat1 and contains residues 198 to 223. It binds GSK-3β in the Axin-binding groove albeit as a helix-turn-helix structure. The N-terminus of the peptide is in close proximity of the pTyr216 side chain. The N-terminal Frat1 helix is not deeply buried in GSK-3β but clamps on to the surface of the protein. The C-terminal helix of Frattide does bind deeply in the Axin-binding groove, with several hydrophobic residues (Leu212, Ile213, Ala216, Val217, and Leu220) anchoring the peptide to GSK-3β. At the N-terminus of the second helix, two hydrogen bonds are formed between GSK-3β side chains and peptide main chain. Tyr288 and Glu290 from GSK-3β interact with the backbone amides of Leu212, Ile213, and Lys214. Lys214 from Frattide also binds the Glu290 carboxylate with its side chain nitrogen. The C-terminal helix-binding groove overlaps with the Axin-binding groove. The tilt of the two helices is slightly different because Frattide is not a straight helix like the Axin peptide, but instead adopts a helix-turn-helix structure. Although the region that facilitates Axin and Frat1 binding is not the substrate-binding groove, the Frat1 peptide selectively inhibits the phosphorylation of Axin and β-catenin, but does not affect the phosphorylation of eIF-2B or glycogen synthase. Similarly GSK-3β complexed with the Frat1 still phosphorylates a CREB-derived peptide substrate [63]. It is important to realize that these studies used peptides. The Frat1 peptide does not bind in the substrate-binding groove where glycogen synthase and eIF-2B interact with GSK-3β. It is possible that the phosphorylation of primed substrates will be inhibited if full-length Frat1 is used. It simply depends on whether bound Frat1 interferes with the binding of these substrates to GSK-3β. Ser9phosphorylated GSK-3β still binds Frat1, but the GSK-3β structures made clear that the N-terminus of the GSK-3β does not stretch to the Axin/Frat1 binding groove [65]. With some substrates there will be interference, and with others not. For instance, overexpressed Frat1 inhibits the phosphorylation of

ACKNOWLEDGMENT

77

the microtubule binding protein, Tau, but does not block the phosphorylation of glycogen synthase by GSK-3β [69]. Both proteins are GSK-3β substrates that undergo primed phosphorylation. A severely truncated form of Xenopus Axin (residue 320 to 429) binds GSK-3β, presumably in the Axin/Frat1 binding groove, and this fragment did not affect the phosphorylation of a glycogen synthase peptide or full-length Tau [70]. It is now clear that GSK-3β phosphorylates many of its substrates via the primed phosphorylation mechanism, and that this phosphorylation mechanism is not pathway speciﬁc. GSK3β has multiple binding sites for protein interactions, and the binding at one site may or may not interfere with the binding and phosphorylation of a substrate at the other site.

4.8

CONCLUDING REMARKS

Advances in the areas of enzymology, molecular biology, and X-ray crystallography have been instrumental in increasing the depth and breadth of our knowledge of kinases. The use of X-ray crystallography has become widespread, and more research teams than ever are practicing this discipline. Certainly X-ray crystallography has served GSK-3β well. The ﬁ rst GSK-3β structures unambiguously explained the primed phosphorylation mechanism and provided insight into the auto-inhibition mechanism of the Ser9 phosphorylated N-terminus. Structures that followed revealed the Axin and Frat1 binding site on the C-terminal domain and explained why the Axin and Frat1 peptides do not necessarily interfere with glycogen synthase phosphorylation. The structures of the GSK-3β–inhibitor complexes have been helpful in unraveling the structure-activity relationships of different chemical classes of kinase inhibitors. GSK-3β is still considered a good target for a number of therapeutic areas, and therefore more crystal structures of small molecule co-complexes can be expected. So far all the GSK-3β–inhibitor complexes published contain inhibitors that bind at the ATP binding site. The unique primed-phosphorylation mechanism provides an additional site for GSK-3 inhibition. For instance, thiadiazolidinones are a class of non-ATP competitive GSK-3β inhibitors that are believed to act in the substrate-binding groove [71]. A structure of GSK3β in complex with a representative of this class might provide valuable insight to leverage this unique binding site to design-speciﬁc inhibitors of GSK-3.

ACKNOWLEDGMENT I would like to thank Marc Jacobs, Subramania Pazhanishamy, Ted Fox, and Mark Namchuk for helpful discussions and review of the article. Portions of this work were conducted within the protein kinase collaboration between Vertex Pharmaceuticals and Novartis Pharma AG.

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5 KINASE–KINASE AND SITE–SITE INTERACTIONS IN THE PHOSPHORYLATION OF TAU BY GSK-3 Khalid Iqbal and Inge Grundke-Iqbal Department of Neurochemistry, New York State Institute for Basic Research, in Developmental Disabilities, Staten Island, New York

Contents 5.1 Introduction 5.2 Modulation of GSK-3 Catalyzed Phosphorylation of Tau by Non–ProlineDependent Protein Kinases In vitro 5.3 Site-Speciﬁc Modulation of Tau Phosphorylation 5.4 Tau Becomes a More Favorable Substrate for GSK-3 When Prephosphorylated by A-Kinase In vivo in Rat Brain

5.1

INTRODUCTION

Discovery of the abnormal hyperphosphorylation of the microtubule associated protein Tau in Alzheimer disease (AD) brain, made in 1986 [1,2], has stimulated enormous interest in protein kinases and phosphatases that regulate the phosphorylation of Tau. One of these protein kinases, which has been

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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most implicated in this Tau pathology, is the glycogen synthase kinase-3 (GSK-3). GSK-3 is constitutively active in resting cells but undergoes rapid inhibition in the presence of insulin/IGF-1, growth factors and other signals [3]. Insulin-stimulated protein kinases phosphorylate GSK-3α at Ser21 and GSK-3β at Ser9, and inactivate both isoforms [4]. Protein kinase B (PKB), which is downstream of phosphotidylinisitol 3kinase (PI3K) inactivates GSK-3 [4,5]. Cleaved protein kinase C delta (PKCδ), during apoptosis, has also been shown to phosphorylate and inactivate GSK-3 [6]. Tau, a phosphoprotein, is a major microtubule associated protein of a normal mature neuron. A major known function of Tau is its interaction with tubulin, the major protein subunit of microtubules. Tau promotes assembly of tubulin into microtubules and helps stabilize the structure of microtubules [7]. These functions of Tau are regulated by its degree of phosphorylation. Normal brain Tau has two to three moles phosphates/mole of the protein, which appears to be optimal for its biological activity. In AD and related tauopathies, tau accumulates in the affected neurons as abnormally hyperphosphorylated protein. In AD brain, Tau is at least three- to four-fold more phosphorylated than in normal adult brain [8]. The abnormally hyperphosphorylated Tau is the major protein subunit of paired helical ﬁ laments (PHF)/ neuroﬁbrillary tangles, a brain histopathological hallmark of AD [1,9]. As much as 40% of the abnormally hyperphosphorylated Tau found in AD brain is cytosolic [8]. Unlike normal Tau, the AD cytosolic abnormally hyperphosphorylated Tau (AD P-tau) does not promote assembly of tubulin into microtubules. Instead of interacting with tubulin, the AD P-tau sequesters normal Tau and as well as the other two microtubule associated proteins, (MAPs) MAP1 and MAP2, and causes inhibition of assembly and disruption of microtubules in vitro [10–14]. This toxic gain of function of AD P-tau is solely due to its abnormal hyperphosphorylation because its dephosphorylation by a protein phosphatase (PP), especially PP-2A, restores its biological activity to a normal state [11,14–16]. The AD abnormally hyperphosphorylated tau and PHF-tau are phosphorylated at over 30 sites, mostly serines/threonines [17,18]. Many of these serines/threonines are followed by prolines, that is, canonical sites for proline-directed protein kinases (PDPKs). Among the three major PDPKs, GSK-3, cdk5, and MAP kinase ERK1/2, GSK-3 can phosphorylate a large majority of these PDPK sites of Tau in vitro and depress its ability to promote assembly of tubulin into microtubules [19–24]. The expression of GSK-3 is high in the adult brain, and it has been found to be associated with neuroﬁbrillary degeneration at all Braak stages of AD [25,26]. Thus a gain of toxic function by abnormal hyperphosphorylation of Tau, and the association of GSK-3 at all stages of neuroﬁbrillary degeneration and its ability to phosphorylate tau at several of the same sites, as seen in AD brain, has drawn a considerable interest in this protein kinase as a therapeutic target for AD and related tauopathies. Normal phosphorylation of tau by GSK-3 has been found to be required for anterograde transport of organelles in cultured PC12

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85

cells [27]. Probably it is the interaction of GSK-3 with other kinases in the phosphorylation of Tau that leads to its pathological phosphorylation. Several studies have demonstrated that the phosphorylation of tau by GSK-3 is modulated by its phosphorylation by other protein kinases, both at common and at noncommon sites. This chapter describes the role of kinase–kinase and site–site interactions involved in the abnormal hyperphosphorylation of Tau by GSK-3.

5.2 MODULATION OF GSK-3 CATALYZED PHOSPHORYLATION OF TAU BY NON–PROLINE-DEPENDENT PROTEIN KINASES IN VITRO Phosphorylation of a protein is a function of not only a protein kinase/s that phosphorylates it but also of both the primary structure and the conformation of the substrate. Phosphorylation of a protein at a site can alter its conformation, which presents it as a different substrate for phosphorylation at an additional site(s) by the same or a different kinase. GSK-3 is known to be especially sensitive to the substrate conformation. The phosphorylation of glycogen synthase [28], the R11 subunit of protein kinase A [29], phosphatase inhibitor-2 [30], and protein phosphatase-1 G subunit [31] by GSK-3 are enhanced when these proteins are ﬁ rst phosphorylated by a different kinase. These types of interactions are especially seen in Tau, which has 80 serines/threonines that can be phosphorylated by serine/ threonine protein kinases. GSK-3 catalyzes the incorporation of nearly 3 mol 32 P into bovine brain Tau in 4 hours at 30˚C (Fig. 5.1A; [23,24]). The ﬁ rst approximately 1.5 mol 32P is incorporated in 1 h, with additional phosphorylation occurring only slowly (∼1.5 mol 32 P/3 h). This rate of phosphorylation is differentially accelerated when Tau is phosphorylated for 1 h at 30˚C by casein kinase (CK)-1, CK-2, A-kinase, C-kinase, calcium, calmodulin-dependent protein kinase II (CaMKII), or Gr kinase (Figs. 5.1B–G; Fig. 5.2A–B). Bovine Tau is already phosphorylated at Ser144 and Ser315. However, this endogenous phosphorylation of bovine Tau does not appear to be involved in the modulation of its phosphorylation by GSK-3, for the dephosphorylated and the endogenously phosphorylated Tau behave similarly in these studies (Fig. 5.2C,D). Unlike A-kinase, Ckinase, and CK-2, prephosphorylation of Tau by CK-1, CaMKII, or Gr kinase does not enhance the subsequent GSK-3-catalyzed phosphorylation. On the contrary, GSK-3-catalyzed 32 P incorporation decreases after prephosphorylation of Tau by CK-1 and CaMKII. These ﬁ ndings suggest that after an initial phosphorylation of Tau by the latter two kinases, it assumes conformations that are unfavorable for subsequent GSK-3catalyzed phosphorylations. CK-2 and C-kinase have been shown to phosphorylate Thr39 [32] and Ser324 (equivalent to Ser313 in bovine Tau) [19]

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A 3 2 1

32

P Incorp. (mol/50,000 gm)

4

0

0

1

2

6

4 3 Time (hrs)

5

6 6 E 4

2

2

P Incorporation (mol/50,000 gm)

4

6 C

4

2

32

32

P Incorporation (mol/50,000 gm)

B

6 D

6

F

4

2

G

6

4

4

2

2

0

0 0

20

40

60 80 Time (min)

100

120

0

20

40

60 80 Time (min)

100

120

in human Tau441. A-Kinase also phosphorylates Ser324, in addition to Ser214, Ser356, Ser407, and Ser416 [33]. Thus it appears that prephosphorylation of Thr39 and Ser324 changes Tau conformation in such a way that additional sites not normally accessible to GSK-3 can now be readily phosphorylated by this kinase. GSK-3 can phosphorylate Ser199, Thr231,

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87

Figure 5.1 Phosphorylation of Tau by GSK-3 and its modulation by other kinases. (A) Time course of phosphorylation of Tau by GSK-3. Tau was phosphorylated by GSK-3, aliquots of the phosphorylation mixture were removed at different times, and the reaction stopped by addition of SDS-stop solution. 32P-labeled Tau was resolved from other proteins by SDS-PAGE, excised from the gels, and 32P incorporation was determined by Cerenkov counting. (B–D) Phosphorylation of Tau by a combination of A-kinase, C-kinase, CK-2, and GSK-3. Tau was separately phosphorylated by A-kinase (Δ, A), C-kinase (••, B), and CK-2 (Ο, C), and aliquots were removed at different times for determination of 32P incorporation. After 60 min (↓), aliquots of the reaction mixtures were separately removed and supplemented with GSK-3 (䉱, •). The incubations were continued and aliquots removed at different times to determine 32 P incorporation into Tau. (E–G) Phosphorylation of Tau by a combination of CK-1, CaM kinase II, Gr kinase, and GSK-3. Tau was separately phosphorylated by CK-1 (Δ, A), CaM kinase II (••, B), and Gr kinase (Ο, C). After 60 min (↓), aliquots of the reaction mixtures were separately removed and supplemented with GSK-3 (䉱, •) and the incubations continued. (Reproduced with permission from Singh et al., 1995, FEBS Lett., 358, 4–8.)

Ser235, Ser396, Ser404, and Ser413 in human Tau441 [22,34]. Phosphorylation of Tau by A-kinase, followed by GSK-3, leads to a marked increase in inhibition of Tau-stimulated microtubule assembly [35]. Recombinant human brain τ3RL (Tau with three repeats and two amino terminal inserts, 3R2N) behaves similarly to bovine Tau, meaning, phosphorylation with A-kinase, C-kinase, CK-1, and CaMKII increase the rate of its phosphorylation by GSK-3 (Fig. 5.2E,F; [23,24]).

5.3 SITE-SPECIFIC MODULATION OF TAU PHOSPHORYLATION The epitopes of various Tau antibodies become good substrates for GSK-3 after Tau is phosphorylated by a non-PDPK. The binding of mAb Tau-1 is known to get inhibited when Tau is phosphorylated at Ser198, Ser199, or Ser202 [1,36]. The binding of mAb Tau-1 is inhibited when Tau is prephosphorylated by PKA or CaMKII before phosphorylation by GSK-3 (Fig. 5.3A–E). Phosphorylation by other kinases, acting singly or in combination, does not signiﬁcantly affect binding of Tau-1. The mAb PHF-1 to pSer396/404, SMI31, and SMI34 to phosphorylated NFH/M that also recognize phosphorylated Tau, and M4 to pThr231/Ser235, recognize only phosphorylated epitopes of Tau [37–39]. PHF-1 recognizes Tau that is phosphorylated by GSK-3 alone, or by a combination of A-kinase + GSK-3, C-kinase + GSK-3,

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CK-1 + GSK-3, and CaMKII + GSK-3. SMI34 recognizes mainly Tau that is phosphorylated by the combined actions of CK-1 and GSK-3. With SMI31, the highest level of binding is achieved when Tau is phosphorylated by a combination of CK1 + GSK-3. Binding of the antibody M4 is promoted when tau is phosphorylated by GSK-3 alone or a combination of CK-1 + GSK-3, and CaMKII + GSK-3. The prephosphorylation of Tau at a speciﬁc site(s) affects not only its phosphorylation by GSK-3 at a particular site(s) but also the rate at which the site-speciﬁc phosphorylation takes place. A signiﬁcant

SITE-SPECIFIC MODULATION OF TAU PHOSPHORYLATION

89

Figure 5.2 Effect of prephosphorylation by non-proline0dependent protein kinases on phosphorylation of Tau by GSK-3. (A, B) Comparison of GSK-3-catalyzed phosphorylations of Tau, which was prephosphorylated by various non-PDPKs. Tau was separately prephosphorylated for 60 min by CK-2 (Δ), C-kinase (•), A-kinase ( ), and CK-1. (䊐), and CaM kinase II ( ), and then used as a substrate for GSK-3. The net 32 P incorporation catalyzed by GSK-3 alone is shown. For comparison, GSK-3 catalyzed phosphorylation of Tau that was not prephosphorylated by any kinase (Ο) is shown. (C, D) Comparison of the phosphorylations of dephosphorylated Tau with Tau not previously dephosphorylated. Tau that was either dephosphorylated (D) with alkaline phosphatase or was not dephosphorylated (C) was used as a substrate for A-kinase (Δ, Ο). After 60 min (↓), aliquots of the reaction mixtures were separately removed and supplemented with GSK-3 (䉱, •). (E, F) Phosphorylation of human tau 39 by different kinases. (E) Comparison of Tau phosphorylation by several kinases. Tau was phosphorylated by A-kinase ( ), C-kinase (•), CaM kinase II (䉱), GSK-3 (Δ), and CK-1 (Ο). (F) Use of different prephosphorylated tau species as substrates for GSK-3. Tau was incubated in the absence (Δ) of presence or A-kinase (.), C-kinase (•), CaM kinase II (䉱), or CK-1 (Ο) and unlabeled ATP. After 2 h at 30˚C, the reactions were stoped by heating at 95˚C for 5 min. After removing denatured kinases (spum at 10,000 × g for 10 min), the reaction mixtures were supplemented with [γ-32P]ATP and GSK-3, and again incubated at 30˚C. In both E and F, aliquots of the reaction mixtures were removed and processed as described. (Reproduced with permission from Singh et al., 1995, FEBS Lett., 358, 4–8.)

decrease in Tau-1 binding occurs only after 21 hours of phosphorylation of Tau either by GSK-3 alone, or a combination of CK-1 + GSK-3 (Fig. 5.3F–I). A decrease in Tau-1 binding is observed after only one hour phosphorylation of Tau by a combination of either A-kinase + GSK-3, or CaMKII + GSK-3. However, whereas A-kinase + GSK-3 catalyzed phosphorylation completely abolishes Tau-1 binding after only three hours, such binding only slowly disappears after phosphorylation of Tau by CaMKII + GSK-3, and is still apparent after 21 hours. Phosphorylation of Tau with GSK-3 alone requires greater than six hours phosphorylation to achieve maximal binding of PHF-1 and SMI31 (Fig. 5.4). However, both of these epitopes are rapidly induced by GSK-3 if Tau is ﬁrst prephosphorylated by A-kinase or CaMKII. Prephosphorylation of Tau by CK-1 rapidly induces the SMI31 epitope, but only slightly improves binding of PHF-1 compared to that promoted by GSK-3 alone. Among the three nonPDPKs (A-kinase, CK-1, and CaMKII), prephosphorylation of Tau by CK-1 and CaMKII are best for rapid induction of the SMI31 and PHF-1 epitopes, respectively, by GSK-3. Both epitopes are maximally induced in about one hour under these circumstances, compared to 21 hours needed for their maximal induction by GSK-3 alone. In only ﬁve minutes, prephosphorylation of Tau by CK-1 (and, to a lesser degree, A-kinase and CaMKII also) induces binding of SMI31 several fold. Under these conditions GSK-3 alone

90

KINASE–KINASE AND SITE–SITE INTERACTIONS

SITE-SPECIFIC MODULATION OF TAU PHOSPHORYLATION

91

Figure 5.3 Pattern of binding of Tau antibodies to different species of phosphorylated Tau. (A–E) Nonphosphorylated Tau (lane 1) or phosphorylated Tau (lanes 2–12) were immunoblotted with Tau-1 (A), PHF-1 (B), SMI34 (C), SMI31 (D), and M4 (E). Tau was phosphorylated by GSK-3 (lane 2), A-kinase (lane 3), A-kinase + GSK-3 (lane 4), C-kinase (lane 5), C-kinase + GSK-3 (lane 6), CK-2 (lane 7), CK-2 + GSK-3 (lane 8), CK-1 (lane 9), CK-1 + GSK-3 (lane 10), CaM kinase II (lane 22), or CaM kinase II + GSK-3 (lane 12). Tau was phosphorylated for 2 h at 30˚C when each kinase was used alone. When a combination of kinases was used, phosphorylation was for 2 h by the ﬁ rst kinase, followed by heating at 95˚C for 5 minutes. The second kinase (GSK-3) was then added and phosphorylation continued at 30˚C for 1 h. In E, Tau was not phosphorylated (lane 1) or phosphorylated by A-kinase + GSK-3 (lane 2), CK-1 + GSK-3 (lane 3), CaM kinase II + GSK-3 (lane 4), or GSK-3 (lane 5). (F–I) Time course of inhibition of Tau-1 binding by different combination of kinases. Tau was not prephosphorylated (A) or was prephosphorylated for 2 h by A-kinase (B), CK-1 (C), and CaM kinase II (D). All samples were then heated at 95˚C for 5 min, transferred back to 30˚C and GSK-3 added to all tubes. Aliquots of the reaction mixtures were then removed at 0 min (lane 1), 5 min (lane 2), 15 min (lane 3), 30 min (lane 4), 1 h (lane 5), 3 h (lane 6), 6 h (lane 7), and 21 h (lane 8). All samples were then immunoblotted with Tau-1. Prephosphorylation of Tau, either by C-kinase or CK-2, did not signiﬁcantly promote inhibition of Tau-1 binding or increase of PHF-1, SMI31, SMI34, M4 binding (below), and these samples were therefore not tested further. (Reproduced with permission from Singh et al., 1995, FEBS Lett., 358, 267–272.)

minimally induces this epitope. A similar conclusion is also true for induction of PHF-1 epitope when Tau is prephosphorylated by CaMKII. In the case of M4 and SMI34, both of these epitopes are maximally induced only after 21 hours of phosphorylation with GSK-3 alone (Fig. 5.4). As shown above in Figure 5.3, recognition of the SMI34 epitope by GSK-3 is promoted when Tau is ﬁ rst prephosphorylated mainly by CK-1. With GSK-3 alone, signiﬁcant binding of SMI34 requires about six hours incubation. After prephosphorylation by CK-1, signiﬁcant binding of this antibody is detected after only ﬁve minutes incubation, but requires nearly six hours to reach a maximum. Prephosphorylation of Tau with either CK-1 or CaMKII increases the rate and extent of binding of M4 to Tau. For this purpose CaMKII is better. Binding of M4 is increased several fold over control after only ﬁve minutes phosphorylation with CaMKII + GSK-3. Maximal binding is achieved in nearly one hour compared to about 21 hours when GSK-3 alone is used. These ﬁndings suggest a possible dual role for the participation of non-PDPKs in the hyperphosphorylation of Tau in an AD-like state. Such hyperphosphorylation is known to occur almost equally at Ser/Thr-Pro and Ser-Thr-X sites [18]. Hence the ﬁ rst possible role of non-PDPKs is to phosphorylate the Ser/Thr-X sites found

92

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TAU BECOMES A MORE FAVORABLE SUBSTRATE

93

Figure 5.4 Time course of site-speciﬁc phosphorylation binding by different combination of kinases. (A–H) Tau that was not prephosphorylated (A, E) or was prephosphorylated by A-kinase (F), CK-1 (C, G), and CaM kinase II (D, H) was then further phosphorylated by GSK-3. Aliquots of the reaction mixtures were removed at different times and immunoblotted either with SMI31 (A–D) or PHF-1 (E–H). Lane numbers and phosphorylation times are the same as in the previous ﬁgure. (I–M) Tau that was not prephosphorylated (I, L) or was prephosphorylated by CK-1 (J, M), and CaM kinase II (K) was then further phosphorylated by GSK-3. Aliquots of the reaction mixtures were removed at different times and immunoblotted either with M4 (IK) or SMI34 (L, M). Lane numbers and phosphorylation times are the same as in previous ﬁgure. (Reproduced with permission from Singh et al., 1995, FEBS Lett., 358, 267–272.)

in PHF-Tau. The second possible role of these kinases is a direct consequence of the ﬁ rst. A major known function of Tau is the stimulation of assembly and maintenance of the structure of microtubules. Phosphorylation of Tau by A-kinase + GSK-3 and CK-1 + GSK-3 inhibits microtubule assembly promoting activity of Tau considerably more than by Tau phosphorylated by GSK-3 alone [35]. Similar effect is seen when the binding of Tau to taxol-stabilized microtubules is studied (Table 5.1). These inhibitory activities of Tau correspond to the phosphorylation of tau induced by GSK-3 in combination with different kinases, which result in select additional sites of phosphorylation (Table 5.2). The phosphorylation of Tau by GSK-3 is also modulated by cdk5, that is, priming of Tau by one PDPK for phosphorylation by a second PDPK [40]. Prephosphorylation of Tau by cdk5 stimulates both the rate and extent of a subsequent phosphorylation catalyzed by GSK-3. Tau modiﬁed by cdk5 is phosphorylated at a threefold higher rate by GSK-3, and the extent of 32P incorporation is increased around 45% compared to the phosphorylation of unmodiﬁed Tau [35]. By contrast, GSK-3-modiﬁed Tau and unmodiﬁed Tau are phosphorylated at approximately the same rate and to the same extent by cdk5. Interestingly prephosphorylation of Tau by GSK-3 does not signiﬁcantly affect its phosphorylation by cdk5.

5.4 TAU BECOMES A MORE FAVORABLE SUBSTRATE FOR GSK-3 WHEN PREPHOSPHORYLATED BY A-KINASE IN VIVO IN RAT BRAIN Forskolin is a well-known speciﬁc activator of A-kinase [41,42]. Phosphorylation of Tau at Ser214 in rat hippocampus increases in a dose-dependent manner after intracerebroventricular (ICV) injection of forskolin [43].

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KINASE–KINASE AND SITE–SITE INTERACTIONS

TABLE 5.1 Binding to microtubules of t3L before and after phosphorylation by different kinases t phosphorylated by

% t bound 94 ± 5.2 87 ± 6.1 86 ± 4.6 81 ± 10.1 61 ± 5.2 50 ± 6.6

None CK-1 GSK-3 A-Kinase CK-1 + GSK-3 A-Kinase + GSK-3

TABLE 5.2 Comparison of t sites phosphorylated by A-kinase, GSK-3, or a combination of the two kinases Kinases

Phosphorylation Sites

A-Kinase

Ser214, Ser324, Ser356, Ser409, Ser416 Ser262, Ser293, Ser305, Ser324, Ser356 Ser235, Ser404 Ser199, Thr231, Ser396, Ser413 Ser181, Ser184, Ser262, Ser400 Ser195, Ser198, Ser202, Thr205, Ser235, Ser262

A-Kinase GSK-3(α) GSK-3(β) GSK-3(α + β) A-Kinase + GSK-3

τ Source

Reference

τ3

Scott et al. [33]

τ4L

Drewes et al. [47]

Bovine Bovine

Yang et al. [22] Ishiguro et al. [21]

τ3L

Wang et al. [35]

τ3L

Wang et al. [35]

Furthermore, under the same conditions, the phosphorylation of Tau at PHF1 site (Ser396/404) increases to several fold, and the level of Tau is decreased where neither serine 198, 199, nor 202 is phosphorylated (Tau-1 site). Forskolin treatment does not affect the total level of Tau as detected by mAb Tau-5. Forskolin treatment increases the A-kinase activity markedly, which correlates with the phosphorylation of Tau at Ser214, Tau-1, and PHF-1 sites. Phosphorylation of tau at Ser214 is catalyzed by A-kinase, while phosphorylation at Tau-1 and PHF-1 sites are catalyzed by GSK-3, cdc-2, cdk5, and MAPK in vitro (for a review, see [44]). However, forskolin treatment does not have any signiﬁcant effect on the activities of GSK-3, cdc2, cdk5, or MAPK (Fig. 5.5). Injection with 100 mM LiCl induces around a 40% reduction in GSK-3 activity in rat hippocampus and has no effect on A-kinase activity (Fig. 5.6). While the phosphorylation of Tau at Ser214 is unaffected, at the PHF-1 and Tau-1 sites the forskolin-induced phosphorylation is

TAU BECOMES A MORE FAVORABLE SUBSTRATE

95

Figure 5.5 Effects of Rp-cAMPS and LiCl on cdc2, cdk5, and MAPK activities. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 100 mM LiCl or 100 μM Rp-cAMPS. The activities of cdc2, cdk5, and MAPK were determined by using speciﬁc peptide substrates. Immunoprecipitation with speciﬁc antibody (for cdk5) was employed to assay the cdk5 activity in the tissue extract. All data are expressed as mean ± SD of eight experiments. (Reproduced with permission from Liu et al., 2004, J. Biol. Chem., 279, 50078–50088.)

dramatically decreased, suggesting that the forskolin-induced phosphorylation of Tau at PHF-1 and Tau-1 sites is catalyzed by basal activity of GSK-3. Co-injection of forskolin with Rp-cAMPS, a speciﬁc PKA inhibitor [45,46], blocks the activation of A-kinase and the phosphorylation of Tau at Ser214, Tau-1, and PHF-1 sites. These ﬁ ndings suggest that the increase in the phosphorylation of Tau at PHF-1 and Tau-1 sites by GSK-3 probably results from the prephosphorylation of Tau at other sites by activation of A-kinase in rat brain. Co-injection of forskolin with PNU112455A (inhibitor of cdc2 and cdk5), PD98059 (inhibitor of MAPK), or SB203580 (inhibitor of p38 MAPK) inhibits the targeted kinase activities but has no effect on GSK-3 activity; nor does the co-injection inhibit the phosphorylation of Tau at Ser214, Tau-1, or PHF-1 site (Figs. 5.7 and 5.8). These studies demonstrate

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TAU BECOMES A MORE FAVORABLE SUBSTRATE

97

Figure 5.6 Effect of LiCl and Rp-cAMPS on the activities of GSK-3 and PKA and phosphorylation of Tau in rat brain hippocampus. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 100 mM LiCl or 100 μM Rp-cAMPS. The activities of GSK-3 and PKA and the phosphorylation state of Tau at various sites in the hippocampus collected 24 h after injection were determined. The activities of GSK-3 (A, H) and PKA (B, G) of the hippocampal extracts were measured using respective speciﬁc peptide substrates. The phosphorylation levels of tau at various sites were determined by western blots using phosphorylation-dependent and site-speciﬁc Tau antibodies PS214 (for Ser214; C and I), PHF-1 (for Ser396 and or -404; D and J), and Tau-1 (for Ser198, -199, and or -202; E and K). The total Tau level was determined by western blots using a phosphorylationindependent tau antibody Tau-5 (F, L). The immunoreactivities (IR) of the Tau staining were quantitated. All data are expressed as mean ± SD of 8 experiments. **p < 0.01 as compared to control injection; ## p < 0.01 as compared to injection with forskolin alone. (Reproduced with permission from Liu et al., 2004, J. Biol. Chem., 279, 50078–50088.)

Figure 5.7 Effects of cdc2/cdk5 inhibitor, PNU 112455A, on activities of GSK-3, PKA, cdc2, and cdk5 and on phosphorylation of Tau in rat hippocampus. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with 200 μM PNU 112455A. The activities of GSK-3 (A), PKA (B), cdc2 (C), and cdk5 (D) were measured by using speciﬁc peptide substrates. The phosphorylation levels of Tau at various sites were determined by western blots using phosphorylation-dependent and site-speciﬁc Tau antibodies PHF-1 (for Ser396 and or -404; E), Tau-1 (for Ser198, -199, and or -202; F), and PS214 (for Ser214; G). The total Tau level was determined by western blots using a phosphorylation-independent Tau antibody Tau-5 (H). Other details are the same as in Figure 5.5. The immunoreactivities (IR) of the tau staining were quantitated. All data are expressed as mean ± SD of 8 experiments. **p < 0.01 as compared to the control injection. (Reproduced with permission from Liu et al., 2004, J. Biol. Chem., 279, 50078–50088.)

98

KINASE–KINASE AND SITE–SITE INTERACTIONS

Figure 5.7 Continued

Figure 5.8 Effects of PD 98059 and SB 203580 on PKA, GSK-3, and MAPK activities and phosphorylation of Tau in rat hippocampus. Rats were injected into lateral ventricle with aCSF as vehicle, 80 μM forskolin alone, or forskolin combined with either 200 μM PD 98059 or 100 μM SB 203580 (inhibitors of MAPK). The activities of PKA (A, B) GSK-3 (C, D), and MAPK (E, F) were assayed by speciﬁc peptide substrates. The phosphorylation levels of Tau at various sites were determined by western blots using phosphorylation-dependent and site-speciﬁc Tau antibodies PHF1 (for Ser396 and or 404; G, H), Tau-1 (for Ser198, -199, and or -202; I, J), and PS214 (for Ser214; K, L). The total Tau level was determined by Western blots using a phosphorylation independent Tau antibody Tau-5 (M, N). The immunoreactivities (IR) of the Tau staining were quantitated. All data are expressed as mean ± SD of 8 experiments. **p< 0.01 as compared to control injection. (Reproduced with permission from Liu et al., 2004, J. Biol. Chem., 279, 50078–50088.)

TAU BECOMES A MORE FAVORABLE SUBSTRATE

Figure 5.8

99

Continued

that the activation of PKA activity by forskolin primes Tau for phosphorylation at Tau-1 and PHF-1 sites by GSK-3. In conclusion, the phosphorylation of Tau by GSK-3 in brain is not solely a function of the level of the activity of this kinase, it is also modulated by its interaction by other protein kinases that phosphorylate it, and as well as by prephosphorylation of Tau at speciﬁc sites, which primes it for subsequent phosphorylation by GSK-3 at other sites.

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ACKNOWLEDGMENTS We thank Janet Murphy and Sonia Warren for secretarial assistance, and Dr. Robert Freedman, GRAMS, for his help with ﬁgures. Studies from our laboratories were supported in part by the New York State Ofﬁce of Mental Retardation and Developmental Disabilities and NIH grant AG019158. REFERENCES 1. Grundke-Iqbal I., Iqbal K., Tung Y.-C., Quinlan M., Wisniewski H.M., Binder L.I. (1986). Abnormal phosphorylation of the microtubule-associated protein (Tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. USA, 83, 4913–4917. 2. Iqbal K., Grundke-Iqbal I., Zaidi T., Merz P.A., Wen G.Y., Shaikh S.S., Wisniewski H.M., Alafuzoff I., Winblad B. (1986). Defective brain microtubule assembly in Alzheimer’s disease. Lancet, 2, 421–426. 3. Cohen P., Alessi D.R., Cross D.A.E. (1997). PDK1, one of the missing links in insulin signal transduction. FEBS Lett., 410, 3–10. 4. Cross D.A., Alessi D.R., Cohen P., Andjelkovich M., Hemmings B. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature, 378, 785–789. 5. Moule S.K., Welsh G.I., Edgell N.J., Foulstone E.J., Pround C.G., Denton R.M. (1997). Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and β-adrenergic agonists in rat epididymal fat cells. J. Biol. Chem., 272, 7713–7719. 6. Tsujio I., Tanaka T., Kudo T., Grundke-Iqbal I., Iqbal K., Takeda M. (2000). Inactivation and phosphorylation of glycogen synthase kinase-3 by protein kinase C in the process of apoptosis induced by inhibition of protein kinase B: Implications on regulation of tau protein phosphorylation. FEBS Lett., 469, 111–117. 7. Weingarten M.D., Lockwood A.H., Hwo S.-Y., Kirschner M.W. (1975). A protein factor essential for microtubule assembly. Proc. Nat. Acad. Sci. USA 72, 1858–1862. 8. Köpke E., Tung Y.-C., Shaikh S., Alonso A. del C., Iqbal K., Grundke-Iqbal I. (1993). Microbutule associated protein Tau: Abnormal phosphorylation of a nonpaired helical ﬁ lament pool in Alzheimer disease J. Biol. Chem., 268, 24374–24384. 9. Grundke-Iqbal I., Iqbal K., Quinlan M., Tung Y.-C., Zaidi M.S., Wisniewski H.M. (1986). Microtubule-associated protein Tau: A component of Alzheimer paired helical ﬁ laments. J. Biol. Chem., 261, 6084–6089. 10. Iqbal K., Zaidi T., Bancher C., Grundke-Iqbal I. (1994). Alzheimer paired helical ﬁ laments: Restoration of the biological activity by dephosphorylation. FEBS Lett., 349, 104–108. 11. Alonso A. del C., Zaidi T., Grundke-Iqbal I., Iqbal K. (1994). Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc. Natl. Acad. Sci. USA, 91, 5562–5566.

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PART II GSK-3 AS A THERAPEUTIC TARGET IN HUMAN PATHOLOGY

6 GSK-3, A KEY PLAYER IN ALZHEIMER’S DISEASE Félix Hernández and Jesús Avila Centro de Biología Molecular “Severo Ochoa” CSIC/UAM, Madrid

Contents 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9

6.1

Introduction GSK-3 Regulation and Alzheimer’s Disease Alzheimer’s Disease, Genetics, GSK-3, and Tau Phosphorylation Tau Phosphorylation by GSK-3 GSK-3 and Other Tauopathies Tau Phosphorylation by GSK-3 and Tau Aggregation Other GSK-3 Substrates Relevant to Alzheimer’s Disease GSK-3 Inhibition as a Therapy for Alzheimer’s Disease Concluding Remarks

INTRODUCTION

Alzheimer’s disease (AD) is a dementia that gives rise to progressive cognitive impairments, including memory failure. Two major neuropathological abnormalities are present in the brain of patients with AD: the extracellular senile plaques (SP) and the intracellular neuroﬁbrillary tangles (NFT). SP are made up of amyloid deposits that are composed of aggregates of βamyloid peptide, a fragment of an integral membrane protein called amyloid Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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precursor protein (APP). Neuroﬁbrillary tangles are aggregates of paired helical ﬁ laments (PHF) whose main component is the hyperphosphorylated form of the microtubule-associated protein called Tau. Now it seems clear that Tau changes in AD are secondary to β-amyloid deposition [1,2] and that Tau is essential for β-amyloid-induced toxicity [3]. However, whether the intracellular connection between β-amyloid and Tau changes in AD is still a matter for debate, and GSK-3β has emerged as one of the possible mediators. In a pioneer study looking for Tau kinases, Ishiguro et al. [4] found a key kinase, associated to microtubules, that was described like Tau kinase I. This kinase was later recognized as GSK-3 [5], a kinase involved in the regulation of glucose metabolism [6]. In vertebrates, two closely related isoforms, GSK3α and GSK-3β are present [7], with GSK-3β being highly expressed in neuronal tissue. Glycogen synthase kinase-3β (GSK-3β) has been proposed as the link between the two neuropathological hallmarks of AD, the extracellular βamyloid and the NFT made of hyperphosphorylated Tau. Deregulation of GSK-3 activity in neurons has been postulated as a key feature in Alzheimer’s disease pathogenesis. Exposure of cortical and hippocampal primary neuronal cultures to β-amyloid induces activation of GSK-3β [8], Tau hyperphosphorylation [9,10], and cell death [9,11]. Blockade of GSK-3β expression by antisense oligonucleotides [11] or its activity by lithium addition [12] inhibits β-amyloid-induced neurodegeneration of cortical and hippocampal primary cultures. Furthermore an interaction of GSK-3β isoform with many of the cellular components related to the neuropathology of AD, such as the APP, the β-amyloid peptide, the metabolic pathway leading to acetylcholine synthesis, the presenilins, which are mutated in many cases of familial AD, and the Tau protein have been described. GSK-3β has been shown to phosphorylate Tau in most sites hyperphosphorylated in NFT both in transfected cells [13] and in vivo [14,15]. Increased levels of active GSK-3β have been found in AD brains and GSK-3β accumulates in the cytoplasm of pre-tangle neurons and can be found associated to NFT [16–18].

6.2

GSK-3 REGULATION AND ALZHEIMER’S DISEASE

Since its discovery, GSK-3 has been found to be involved in many physiological process. GSK-3 plays important roles in embryonic development, cell differentiation, microtubule dynamics, cell cycle division, cell adhesion, glucose metabolism, and apoptosis [19,20]. In addition, a growing amount of experimental data has demonstrated its involvement in some pathological processes as Alzheimer’s disease. The GSK-3 protein (E.C. 2.7.1.37) is a proline-directed serine/threonine protein kinase that was originally identiﬁed and named for its ability to phosphorylate, among other kinases, the enzyme glycogen synthase. The CNS is

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the tissue with the highest GSK-3 level [7]. In mammals two GSK-3 isoenzymes (α and β) have been described [7]. They share the 95% amino acid identity of having the N-terminal and C-terminal ends the regions with less homology. Both isoenzymes are the product of two independent genes. GSK3α gene encodes a 51 kDa protein, while GSK-3β gene encodes a 47 kDa protein (mapped to chromosome 19q12.3 and 3q13.3 respectively [21,22]). A new alternative splicing isoform of GSK-3β with an additional 13 aa insert (exon 8A) in the catalytic domain has been described [23]. The GSK-3β gene has been analyzed [24] and its promoter studied in order to identify variations that could be associated with an abnormal function [25]. However, these studies have concluded that aberrant Tau phosphorylation is not due to mutations in the GSK-3 beta promoter. GSK-3 is regulated at several levels. Activation of Tyr216 by phosphorylation in GSK-3β or Tyr276 in GSK-3α is necessary for its activity [26]. Mutation of that residue reduces the activity [26]. From crystal structure it has been proposed that unphosphorylated Tyr216/276 blocks the access of primed substrates. The structure of phosphorylated GSK-3β has been published [27] showing that phosphorylated Tyr216 suffers a conformational change that allow the primed substrate to bind the enzyme. Which is the kinase that phosphorylated Tyr216? In Dictyostelium the Zaphod kinase activates GSK-3 by Tyr-phosphorylation [28]. In mammalian, experimental data suggest that Fyn tyrosine kinase [29] or some related tyrosine kinase are involved. However, it seems that phoshorylation in that residue is result of an autophosphorylation event [30]. In postmortem samples from AD patients active GSK3β co-localize with hyperhosphorylated Tau [31]. To support these data an accumulation of active GSK-3β has been observed in dystrophic neuritis close to amyloid deposit in transgenic mice overxpressing mutant APP [32]. Inhibition of the enzyme is mediated by two different types of mechanisms [19]. The ﬁ rst mechanism is used by insulin and growth factors and is mediated mainly by PKB, which phosphorylates Ser9 in GSK-3β and Ser21 in GSK-3α. Structural studies have suggested a model for the inhibition [33,34]. Thus the phosphorylated Ser9/21 binds as a competitive pseudosubstrate to the primed-binding site inhibiting the binding of the protein to be phosphorylated. In this way it has been suggested that β-amyloid could facilitate the phosphorylation of Tau protein as β-amyloid could act as an antagonist of the insulin receptor [35], facilitating the activation of GSK-3 through inhibition of Ser9/21 phosphorylation. Interestingly GSK-3β phosphorylated at Ser9, which inactivates GSK-3, is found in the majority of neurons with neuroﬁbrillary tangles and dystrophic neurites of senile plaques in AD and other phospho-Tau-containing neurons in other tauopathies (for a review, see [36]). The second mechanism of GSK-3 inhibition is used by the Wingless (Wnt) signaling pathway. GSK-3 is present in a multiprotein complex formed by axin and APC. GSK-3 is able to phosphorylates β-catenin, targeting it for proteasome degradation [37]. Wnt proteins bind to the Frizzled receptor activating

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the Disheveled protein. This inhibits GSK-3 activity by disrupting the multiprotein complex. As a consequence β-catenin is accumulated and translocated into the nucleus where it activates the transcription through binding to transcription factors. It has been described that mutations in PS-1 may potentiate neuronal apoptosis through β-catenin destabilization [38,39]. Interestingly an association of PS-1 with GSK-3β and Tau protein has been described [40], and transgenic mice overexpressing GSK-3β present a decrease in nuclear β-catenin levels [41]. If β-catenin modulates gene expression, the consequences of the altered levels of β-catenin may be an important factor in the onset of AD.

6.3 ALZHEIMER’S DISEASE, GENETICS, GSK-3, AND TAU PHOSPHORYLATION AD has been classiﬁed in two types: AD of familiar origin (FAD, monogenic) or sporadic AD (polygenic). Three genes have been found to be the cause of the onset of FAD. The ﬁ rst one that was described codiﬁed for APP, and the other two for two proteins known as presenilin-1 (PS-1) and presenilin-2 (PS2). GSK-3 is involved in APP processing. Thus GSK-3β phosphorylates APP, increasing its intracellular processing [42,43]. Furthermore inhibition of GSK-3β reduces the production of β-amyloid [44,45]. One of the functions of PS-1 and PS-2, the other proteins linked to FAD, is to facilitate the cleavage of APP yielding β-amyloid [46,47]. Additionally it was found that aggregates of β-amyloid induce Tau phosphorylation [9] and that GSK-3β (Tau kinase I) was essential for the β-amyloid induced neurotoxicity [11]. These, and other studies, were the base of the so-called amyloid hypothesis [48,49]. This hypothesis suggested that the origin of AD must be in the appearance of βamyloid peptide. β-Amyloid production is facilitated by mutations associated to FAD in APP or PS (a gain of function). β-Amyloid will aggregate, and the aggregates facilitate Tau phosphorylation; as consequence of that phosphorylation, Tau protein polymerizes into PHF and later aggregates into NFT. If that is the case, PS mutations yielding increasing amounts of β-amyloid peptide will induce a faster onset of FAD. However, it is not the case [50] as the amount of β-amyloid generated in cells transfected with different PS-1 mutations did not correlate with the starting age of FAD caused by that mutation. To support this view, a novel PS1 mutation has been recently described in a patient with Pick-type tauopathy whithout extracellular β-amyloid deposits [51]. Thus preselinin proteins could have another function that could be important for AD onset. One of these functions was described by Weihl et al. [52], indicating that PS-1 downregulates PKB. Afterward Baki et al. [53] described that PS-1 activates PI3K-inhibiting GSK-3 activity and Tau hyperphosphorylation. Therefore it is possible that some PS-1 mutations result in a lack of PI3K activation (loss of function), which results in Tau phosphorylation. To resume, it can be speculated that there exist two path-

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ways from preselinin to induce AD. In one case, a gain of function results in the appearance of β-amyloid and, afterward, Tau phosphorylation by GSK-3; in the other case, a loss of function results but also Tau phosphorylation by GSK-3. Thus a common feature for FAD due to PS-1 mutations is the appearance of Tau phosphorylated by GSK-3.

6.4

TAU PHOSPHORYLATION BY GSK-3

Phosphorylation of Tau has been proposed as an important nongenetic factor involved in tauopathies as AD. In addition to the residues present in the microtubule binding domain (MBD), Tau molecules present in PHFs are hyperphosphorylated at sites ﬂanking the MBD. More than 30 sites could be modiﬁed by phosphorylation in Tau protein. These sites have been divided in nonproline kinase directed sites and proline kinase directed sites [54] with mainly the second class being preferentially modiﬁed by GSK-3. Many GSK-3 substrates require a priming phosphorylation by a priming kinase on a Ser or Thr residue with four amino acids at the C-terminal to the phosphorylatable Ser or Thr residue. The crystal structure of human GSK-3β has provided a model for the binding of prephosphorylated substrates to the kinase (PDB ID are 1I09 [33] and 1H8F [34]). Thus, primed Ser/Thr are recognized by a positively charged “binding pocket” that facilates the binding of primed substrates. Nevertheless, the GSK-3 phosphosites on Tau molecule have been further divided in primed and nonprimed sites [55], depending on the requirement (or not) of a previous modiﬁcation, by other kinases, at the residue located in position +4 of that which is afterward modiﬁed by GSK-3 [6]. Phospho-Tau isolated from the brain of Alzheimer’s disease patients contain more primed than unprimed GSK-3 modiﬁed sites [55]. Several of these sites have been characterized, and antibodies that speciﬁcally recognize those phosphosites have been generated. In the case of primed sites, the knowledge of the priming kinase is of obvious interest. Kinases like cdk-5 [56,57], PAR-1 [58], or PK-A [59] could act like priming kinases for a further GSK-3 phosphorylation. GSK-3β can also be responsible of Tau phosphorylation in epitopes that are phosphorylated by other kinases. Thus it has been recently reported that in transgenic mice overexpressing FTDP-17 Tau and GSK-3β one of the main epitopes phosphorylated is the one recognized by the antibody 12E8 (Ser262/356) [60]. This is in agreement with the fact that 12E8 labeling of AD brains predominates in tangles when compared with other phosphoepitopes [61]. However, Ser262/356 epitope has not been described to be phosphorylated by GSK-3. Drosophila PAR-1 kinase, and likely its mammalian homologue MARK, phosphorylate both serines [58], generating a primed residues to create a GSK-3 consensus sequence. Thus it can be speculated that Tau protein when phosphorylated by GSK-3 at, for example, AT-8 and/or PHF-1 epitopes that ﬂank MBD, has less afﬁ nity for microtubules and therefore the 12E8 epitope is then accessible to be phosphorylated by Ser262/356 kinases.

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The contribution of α- and β-isoforms need further analysis, and it is necessary to understand the contribution of both isoforms to Tau phosphorylation. In fact, the recently described new splicing β-isoform has less afﬁ nity for Tau protein than the low molecular weight β-isoform [23]. The contribution of axonal and/or somatodendritic GSK-3 pools to generate hyperphosphorylated Tau has to be also clariﬁed. Thus it has been recently published that inactive GSK-3β is enriched in the growth cone of the axons [62], showing that there exist a GSK-3 neuronal gradient. In the same way phospho-Tau epitopes are not homogeneously distributed, and there exist gradients in cultured neurons [63]. Thus it is well established that there exist a gradient of Tau-1 epitope (an antibody that recognize unphosphorylated Ser199/202) through the axon, being the signal more intense in the distal part of the axon than in the beginning of the same. In the same way it is clear that Tau protein is phosphorylated in AT-8 epitope (an antibody that recognizes phospho Ser202) in the proximal axon. Understanding how phosphogradients are formed can help us to understand how Tau can be aberrantly hyperphosphorylated.

6.5 GSK-3 AND OTHER TAUOPATHIES Mutations in Tau protein have not been described in AD. However, several Tau mutations have been found in another type of tauopathy as is FTDP-17 [64–66]. Several of these mutations are mapped within the microtubulebinding repeats or close to them, and as a consequence a partial loss of microtubule-binding function has been reported, providing a mechanism by which unbound Tau might be more prone to accumulate and thus facilitate aggregation [67,68]. Taking into account that, as we have mentioned before, missence mutations have not been described in AD, a likely hypothesis is that aberrant phosphorylation can imitate mutations found in FTDP17 patients. Interestingly FTDP-17 mutations are concentrated in the Cterminal end and aberrant phosphorylation, as that generated by GSK-3β, is located in the same region. However, GSK-3β is able to exacerbate FTDP-17 Tau pathology at least in transgenic mice. Thus a transgenic mice overexpressing the enzyme GSK-3β and Tau protein with a triple FTDP-17 mutation show an increase in Tau phosphorylation/aggregation, demonstrating that both types of Tau modiﬁcations can act synergistically to induce neuropathology [60]. A similar approach has been performed in lower eukaryotes, such as the fruit ﬂy, to analyze the mechanisms by which GSK-3 and Tau are able to contribute to neurodegeneration. Thus overexpression of human wild-type Tau and shaggy/zeste white-3, the homologue of human GSK-3β in transgenic ﬂ ies has also been investigated [69]. In these ﬂ ies neurodegeneration was exacerbated and abnormal ﬁ laments were observed, thus strengthening the importance of GSK-3 in this process. Interestingly transgenic ﬂ ies expressing human wild-type and mutant Tau (R406W) both die

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prematurely [70], although the toxicity of mutant Tau was higher than that of wild-type Tau. In the majority of FTDP-17 victims, the polymers assembled from Tau only contain the 4-repeat isoforms [71] and in several FTDP-17 families, the only Tau mutations found have been those that affect the splicing of exon 10, which increases the ratio of 4-repeat respect to 3-repeat isoforms [71]. Taken together, these observations indicate that the 4-repeat forms of Tau favor ﬁbril formation compared to the 3-repeat forms. In this context it has been described that GSK-3β is able to regulated Tau splicing through SC35 protein, a member of the SR family of splicing factors [72]. Progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, and Pick’s disease are other tauopathies with altered Tau metabolism whose main neuropathological characteristic is the presence of hyperphosphorylated Tau protein in the absence of amyloid deposits (for a review, see [73,74]). These Tauopathies differ in the levels of Tau phosphorylation as well as in the Tau isoforms involved in aberrant aggregation. However, although no differences in the expression of GSK-3 have been described, GSK-3 was enriched in sarcosyl-insoluble fractions obtained from these tauopathies, suggesting association of this kinase with Tau hyperphosphorylation in Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration [75]. Interestingly GSK-3β phosphorylated at Ser9, which inactivates GSK-3, is found in the majority of neurons with neuroﬁbrillary tangles and dystrophic neurites of senile plaques in AD, and in Pick bodies and other phospho-Tau-containing neurons and glial cells in other tauopathies (for a review, see [36]).

6.6 TAU PHOSPHORYLATION BY GSK-3 AND TAU AGGREGATION The role of phosphorylation in the self-assembly of Tau is a fundamental question in the study of AD and other Tauopathies. It has been suggested that phosphorylation of some speciﬁc Tau sites may be a prerequisite for its assembly [76,77]. GSK-3β is one of the best candidate enzymes for generating the hyperphosphorylated Tau that is characteristic of PHFs. Transgenic animals overexpressing GSK-3β have AD-hyperphosphorylated Tau, although no ﬁ laments could be detected [41]. However, when the same animals were crossed with transgenic mice overexpressing a FTDP-17 Tau, isoform ﬁ laments were obtained showing that GSK-3β is involved in aberrant Tau aggregation [60]. However, in vitro experiments to test the effect of phosphorylation on Tau self-assembly have shown varied results, presumably reﬂecting slight differences in experimental conditions. In some experiments phosphorylated Tau displays a decreased propensity to aggregate [78], whereas in other conditions the aggregation propensity increases [79]. That discrepancy in the results can be due to differences in Tau assembly inducers or in the status of Tau

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phosphorylation. Probably phosphorylation of some regions even inhibits aggregation, while phosphorylation of other regions induces Tau polymerization. Compounds such as HNE [79] and several quinones [80] catalyze the formation of ﬁbrillar aggregates of phosphorylated Tau peptides, but they fail to have an observable effect if the peptides are in a nonphosphorylated form. Another fundamental question refers to the molecular structure of Tau protein in the PHF particle. The current view is that pathological protein aggregation must involve formation of β-sheet structure giving rise to the typical amyloid ﬁbrils [81]. The discovery of α-helix structure in puriﬁed PHFs [82] indicates that PHFs are not a typical case of amyloidosis. The role of α-helix structure in PHF formation is also supported by data showing that the region implicated in forming the core of PHFs (i.e., MBD domain) becomes very α-helical upon addition of the helix-promoting agent TFE [83–85]. As said before, the fact that phosphorylation of Tau occurs in sites of the same region that is involved in forming the core of PHFs has led to suggestions that phosphorylation controls assembly. A mechanistic model of the interplay between phosphorylation and assembly has been proposed based in experimental data obtained with a peptide corresponding to Tau region 317–335. PKA is able to phosphorylate serine 324 [86] and then generate a consensus site recognize by GSK-3, which is phosphorylated by GSK-3β [87]. The biphosphorylated peptide has a higher propensity to form α-helical structures than the unphosphorylated one [87]. The amphipathic α-helix generated suggests a mechanism for polymerization in which the interaction between two amphipathic α-helices is the ﬁ rst step in polymerization [83,87]. It can be matter of discussion if, after the interaction of the phosphorylated peptide, PHF formation is facilitated in vivo by some of the compounds that induce Tau assembly in vitro, such as quinones and HNE

6.7 OTHER GSK-3 SUBSTRATES RELEVANT TO ALZHEIMER’S DISEASE GSK-3 is a multifunctional kinase that phosphorylates cytoskeletal proteins, metabolic enzymes and transcription factors. Thus GSK-3 deregulation may cause neurodegeneration by several pathways in addition to Tau hyperphosphorylation. Some of these proteins (i.e., β-catenin, APP, PS-1 and 2) have been already discussed. However, there are other GSK-3 substrates that can be also related to neurodegeneration. Thus pyruvate dehydrogenase when phosphorylated by GSK-3 is known to lead to mitochondrial dysfunction [88]. Inhibition of PDH by GSK-3 may alter energy metabolism and acetylcholine synthesis, an abnormality present in the brain of patients with AD. It can be speculated that if GSK-3β is altered in AD, proteins phosphorylated by the kinase that control the establishment and maintenance of neuronal polarity may be important in the neurodegenerative disease.

GSK-3 INHIBITION AS A THERAPY FOR ALZHEIMER’S DISEASE

115

Thus Tau protein can be one of these protein as well as another microtubule binding proteins as MAP-1B [89] and MAP-2 [90]. Another important target of GSK-3 is the protein called collapsing response mediator protein-2 (CRMP2), a protein that in a nonphosphorylated form is enriched in the growth cone of the axon and that has been involved in the establishment of neuronal polarity through its tubulin binding activity [91].

6.8 GSK-3 INHIBITION AS A THERAPY FOR ALZHEIMER’S DISEASE All the data discussed above raise the possibility of designing novel therapeutic interventions aimed at blocking GSK-3 as an enzyme that promotes Tau aggregation, thus ameliorating neurodegeneration in Tauopathies. Lithium, a relatively selective GSK-3 inhibitor [92], is the main inhibitor used to implicate GSK-3 in a process. Lithium is a noncompetitive inhibitor with respect to the two GSK-3 substrates, ATP, and the substrate to phosphorylate. On the other hand, kinetic studies have demonstrated that lithium is a competitive inhibitor with respect to Mg2+ [93], suggesting that there exist two Mg 2+ binding sites: one Li-sensitive (as well as to other cations of similar ionic radii like beryllium) and the other Li-insensitive involved in ATP complex [94]. Thiadiazolidinones are able to inhibit GSK-3 in a non-ATP competitive manner [95] while indirubins [96], maleimides [97], and thiazoles [98] are analogues of ATP that bind to GSK-3 beta’s ATP binding pocket. Previous studies have suggested that lithium, a widely used drug for affective disorders that inhibits GSK-3 at therapeutically relevant concentrations, may be a useful drug for modulating Tau hyperphosphorylation and neurodegeneration in AD. First, lithium treatment has been demonstrated to inhibit Tau phosphorylation in both cultured neuronal cells and in vivo in rat brain [15,99]. Second, lithium also blocks Tau hyperphosphorylation and inhibits cell death in cultured neurons treated with β-amyloid peptide [12]. Third, lithium seems to provide neuroprotection against a variety of toxic insults in both cultured neurons and in vivo in several animal models [100,101]. Fourth, some evidence from brain-imaging studies is consistent with the neurotrophic/ neuroprotective effect of chronic lithium treatment in patients with bipolar mood disorder [102]. As a ﬁ nal point, the protective effect of lithium in several animal models has been tested. Thus the ﬁ rst reported transgenic mouse in which lithium has been analyzed was an animal model expressing human Tau with three FTDP-17 missense mutations: G272V, P301L, and R406W [103]. Chronic lithium treatment decreased the formation of the aberrant Tau aggregates [104]. The same results were obtained in another animal model expressing human Tau with the P301L mutation [105]. In this second paradigm a selective GSK-3 inhibitor from AstraZeneca [98] was also tested with similar results. All these data support the idea that GSK-3 inhibitors can be beneﬁcial to prevent Tau pathology or, although not tested, even reversal

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of neuropathology as has been described for another neuropathologies [106]. Further it has been recently described in mice expressing in a conditional manner (Tet-on system) human FTDP-17 Tau isoform that after the suppression of transgenic Tau with doxicicline, memory function recovered, and neuron numbers stabilized, although NFTs did not reverse [107].

6.9

CONCLUDING REMARKS

A growing amount of data support the hypothesis that GSK-3 in vivo may contribute to AD. Thus GSK-3 deregulation induces a severe Tau pathology and interfere with the metabolism of β-amyloid, the other hallmark of AD (Fig. 6.1). Given the signiﬁcant role of GSK-3 in a variety of effects linked to mechanisms related to AD, GSK-3 inhibition using lithium or small molecule inhibitors is a promising hypothesis for AD and related Tauopathies.

PS-1+APP Aß

GSK-3ß P -Tau

Aggregation

SP NFT Figure 6.1 Interaction of GSK-3 with the cellular components related to the neuropathology of AD, such as the amyloid precursor protein, the β-amyloid peptide, the presenilins and Tau protein. See color plates.

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GSK-3, A KEY PLAYER IN ALZHEIMER’S DISEASE

(2001). Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal Tau phosphorylation in Alzheimer’s disease: A property common to most cyclin-dependent kinase inhibitors? J. Biol. Chem., 276, 251–260. Coghlan M.P., Culbert A.A., Cross D.A., Corcoran S.L., Yates J.W., Pearce N.J., Rausch O.L., Murphy G.J., Carter P.S., Roxbee Cox L., Mills D., Brown M.J., Haigh D., Ward R.W., Smith D.G., Murray K.J., Reith A.D., Holder J.C. (2000). Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol., 7, 793–803. Bhat R., Xue Y., Berg S., Hellberg S., Ormo M., Nilsson Y., Radesater A.C., Jerning E., Markgren P.O., Borgegard T., Nylof M., Gimenez-Cassina A., Hernandez F., Lucas J.J., Diaz-Nido J., Avila J. (2003). Structural insights and biological effects of glycogen synthase kinase 3-speciﬁc inhibitor AR-A014418. J. Biol. Chem., 278, 45937–45945. Munoz-Montano J.R., Moreno F.J., Avila J., DiazNido J. (1999). Downregulation of glycogen synthase kinase-3 beta (GSK-3 beta) protein expression during neuroblastoma IMR-32 cell differentiation. J. Neurosci. Res., 55, 278–285. Chen R.W., Chuang D.M. (1999). Long term lithium treatment suppresses p53 and Bax expression but increases Bcl-2 expression. A prominent role in neuroprotection against excitotoxicity. J. Biol. Chem., 274, 6039–6042. King T.D., Bijur G.N., Jope R.S. (2001). Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res., 919, 106–114. Moore G.J., Bebchuk J.M., Wilds I.B., Chen G., Manji H.K. (2000). Lithiuminduced increase in human brain grey matter. Lancet, 356, 1241–1242. Lim F., Hernandez F., Lucas J.J., Gomez-Ramos P., Moran M.A., Avila J. (2001). FTDP-17 mutations in Tau transgenic mice provoke lysosomal abnormalities and Tau ﬁ laments in forebrain. Mol. Cell. Neurosci., 18, 702–714. Perez M., Hernandez F., Lim F., Díaz-Nido J., Avila J. (2003). Chronic lithium treatment decreases mutant Tau protein aggregation in a transgenic mouse model. J. Alzheimers Dis., 5, 301–308. Noble W., Planel E., Zehr C., Olm V., Meyerson J., Suleman F., Gaynor K., Wang L., LaFrancois J., Feinstein B., Burns M., Krishnamurthy P., Wen Y., Bhat R., Lewis J., Dickson D., Duff K. (2005). Inhibition of glycogen synthase kinase-3 by lithium correlates with reduced tauopathy and degeneration in vivo. Proc. Natl. Acad. Sci. USA, 102, 6990–6995. Yamamoto A., Lucas J.J., Hen R. (2000). Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell, 101, 57–66. Santacruz K., Lewis J., Spires T., Paulson J., Kotilinek L., Ingelsson M., Guimaraes A., DeTure M., Ramsden M., McGowan E., Forster C., Yue M., Orne J., Janus C., Mariash A., Kuskowski M., Hyman B., Hutton M., Ashe K.H. (2005). Tau suppression in a neurodegenerative mouse model improves memory function. Science, 309, 476–481.

7 GLYCOGEN SYNTHASE KINASE 3: A TARGET FOR NOVEL MOOD DISORDER TREATMENTS Todd D. Gould and Husseini K. Manji Laboratory of Molecular Pathophysiology NIMH, NIH, Bethesda, MD

Contents 7.1 7.2 7.3 7.4

Introduction to Mood Disorders and Limitations of Current Treatments Lithium Salts as Medication Molecular Effects of Lithium A Newer Target of Lithium: Direct and Indirect Inhibition of Glycogen Synthase Kinase 3 7.5 Effects of Other Thymoleptic Medications on GSK-3 Mediated Signaling 7.5.1 Valproate 7.5.2 Antidepressants 7.5.3 Electroconvulsive Seizures 7.5.4 Antipsychotics 7.5.5 Estrogen 7.6 Support from Studies of Human Genetics and Rodent Behavior 7.6.1 Genetics 7.6.2 Rodent Behavioral Models 7.7 Concluding Remarks

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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7.1 INTRODUCTION TO MOOD DISORDERS AND LIMITATIONS OF CURRENT TREATMENTS Bipolar disorder and unipolar depression are classiﬁed as mood disorders. They are common, severe, and chronic illnesses. Depression is typiﬁed by a depressed mood, anhedonia (inability to experience pleasure), feelings of worthlessness or excessive guilt, impaired sleep (either insomnia or hypersomnia), cognitive and concentration deﬁcits, psychomotor changes, recurrent thoughts of death or suicide, and a variety of neurovegetative symptoms. In bipolar disorder, patients typically alternate (albeit not in a one-to-one manner) between episodes of depression (mostly indistinguishable from unipolar depression) and episodes of mania, which is characterized by a heightened mood, hyperaroused state, racing thoughts, increased speed and volume of speech, quicker thought, brisker physical and mental activity levels, inﬂated self-esteem, grandiosity, increased energy (with a corresponding decreased need for sleep), irritability, impaired judgment, heightened sexuality, and sometimes frank psychotic symptoms such as hallucinations and delusions. Bipolar Disorder is further classiﬁed into bipolar I or II, based on clinical presentation. Bipolar I disorder is deﬁ ned by a history of one or more manic episodes. Bipolar II is deﬁ ned by a history of one or more hypomanic episodes and at least one depressive episode. In hypomania, a less severe form of mania, the changes noted for the description of mania above are generally observed but are more moderate and do not result in hospitalization. Both diseases commonly ﬁ rst appear in young adulthood, though early and late onset of both diseases is not uncommon. They are often comorbid with other conditions, most commonly anxiety disorders with depression and drug and alcohol abuse with bipolar disorder. The courses of both depression, bipolar I, or bipolar II are characterized by episodes of mood changes, separated by periods of euthymia (normal mood). However, their episodic courses with intervening periods of recovery belie the severe impact of these diseases. The accumulating effect of recurring bouts of depression and/or mania leads to an increased rate of marital and family breakdown, unemployment, impaired career progress, and consequent ﬁ nancial difﬁculties. Both diseases are pervasive, being associated with increased morbidity and mortality. This arises not only from a rate of suicide approaching 15% for both disorders, but additionally the existence of signiﬁcant medical comorbidities, often limited social and economic functioning, and poor inter-episode recovery: aspects that are increasingly being recognized (see [1] and accompanying special issue of Biological Psychiatry). For example, the World Health Organization ranks diseases not solely based on fatal outcomes, but additionally nonfatal outcomes in a measurement referred to as a disability-adjusted life year (DALY) [2]. By this metric they found in 1999 that worldwide, depression ranked the ﬁ fth worst and bipolar disorder

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ranked twentieth. Further DALYs from depression and bipolar disorder diseases were higher in developed countries such as the United States. The World Health Organization projects that by the year 2020 depression will be the second cause of DALYs worldwide (only to cardiovascular disease) [2]. While once thought to have primarily environmental origins, uncontroversial evidence now exists that genetic and other biological factors play a central role in the pathogenesis of mood disorders. Epidemiological evidence consisting of studies involving families, siblings, and twins is supportive of a strong genetic diathesis. For instance, while bipolar disorder affects about 1% of the world’s population overall, monozygotic twins and ﬁ rst-degree relatives are afﬂ icted about 60% and 7% of the time, respectively [3,4]. Major depression is even more common than bipolar disorder in the overall population (15% lifetime risk); however, ﬁrst-degree relatives of probands develop the disease at three times this rate [5,6]. Heritability estimates for bipolar disorder and depression are approximately 80% and 33%, respectively [5,7,8] (compare to type 2 diabetes at 26% [9]). While bipolar disorder is equally prevalent in men and women, depression is at least twice as common in women. Genetic linkage and association studies are beginning to show promise but are hindered by phenotypic and genetic heterogeneity, in addition to the likely complex contribution of epigenetic and environmental factors [10–12]. However, the true pathology of these diseases has proved elusive. Past theories have centered on dysregulation of monoaminergic (serotonin and noradrenergic) signaling pathways, which are involved in the actions of some medications (vide infra) and alteration of which can precipitate mood episodes (e.g., reserpine precipitating depression and amphetamine or l-dopa induced mania). Updated theories take into account changes in neuronal plasticity predisposing neuron and glial cells of the brain to impaired function [13]. There exist varied treatments for mood disorders. Medications useful for treating mania and depression fall into two general classes; these are mood stabilizing medications and antidepressants, respectively. Lithium was the ﬁ rst treatment speciﬁcally for bipolar disorder, and it has proven efﬁcacy in the treatment of both bipolar mania and depression [14]. Additional classes of medications that have been successful in double-blind studies for the treatment of bipolar disorder include select anticonvulsants (valproate, carbamazepine, and lamotrigine), and antipsychotics. The antipsychotic class includes the atypicals (e.g., clozapine, olanzapine, riperidone) and typicals (haloperidol, chlorpromazine), and while they appear most efﬁcacious in the treatment of mania, some studies suggest efﬁcacy in the treatment of depression as well [15,16]. Unipolar and bipolar depression is generally treated with antidepressants belonging to medication classes that act by increasing synaptic levels of monoamines (generally serotonin and/or norepinephrine). These

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medications include tricyclics (e.g., desipramine, imipramine), selective serotonin reuptake inhibitors (SSRIs; ﬂuoxetine, paroxetine), and monoamine oxidase inhibitors (MAOIs; phenelzine, tranylcypromine). Refractory depression, and also mania, may be treated with electroconvulsive therapy (ECT). However, despite these options there exists an urgent need for novel medications for the treatment of both bipolar disorder and depression. Current compounds often do not bring about full remission, and if they do, it is after a minimum of many weeks, if not months of treatment. Additionally they often lack the complete ability to prevent breakthrough episodes, lack speciﬁcity because of disease heterogeneity, or commonly have undesirable side effects even in the best efﬁcacy scenarios (e.g., sexual side effects with SSRIs [17]). The development of novel therapeutics for severe mood disorders has been hindered by both limited knowledge regarding the underlying neurobiology of the disorders and limited evidence regarding how the most useful medications actually exert their beneﬁcial effects [18–20]. Without a ﬁ rm understanding of these issues, it will be impossible to develop truly novel therapeutics for the treatment of mood disorders. Thus ongoing studies to elucidate both the complex etiologies of these disorders and the relevant mechanism of action (both direct and downstream) of current drugs hold vast promise for major advances. As mentioned, almost all current antidepressants (tricyclics, SSRIs, MAOIs, etc.) appear to initially target neural circuits that rely upon the neurotransmitters serotonin and norepinephrine. In contrast, the initial relevant target(s) of mood-stabilizing agents (e.g., lithium and valproate) are not established, but there is evidence for direct inhibition of particular intracellular enzyme(s) (see [18] for review). Importantly, a great deal of experimental evidence suggests that the downstream therapeutic targets of both antidepressants and mood stabilizers are critical intracellular signaling pathways [10]. Thus a simple increase in the intrasynaptic levels of a “deﬁcient neurotransmitter” likely does not explain the true mechanisms of action of antidepressants. Indeed, this seems unlikely since the therapeutic effects are only observed after weeks of administration. This observation, coupled with a number of negative and spurious ﬁ ndings, has led to the conclusion that increasing intrasynaptic levels of serotonin and/or norepinephrine is simply an initiating event that induces a cascade of signaling and gene expression changes in critical neuronal circuits, effects that are ultimately responsible for the medications’ therapeutic effects. It is unfortunate that most recent pharmaceutical efforts to develop “novel” medications for the treatment of bipolar disorder have relied primarily on testing medications initially envisioned and approved for other indications (most generally antipsychotic and antiepileptic drugs for the treatment of bipolar disorder) in hope that they may have efﬁcacy. An alternative approach is to develop medications speciﬁcally for bipolar disorder based on direct and indirect targets of known medications [18].

MOLECULAR EFFECTS OF LITHIUM

7.2

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LITHIUM SALTS AS MEDICATION

As mentioned, lithium represents a common treatment for bipolar disorder. Lithium salts were ﬁ rst used therapeutically in the nineteenth century as remedies for sleeplessness and gout. Since the report of their efﬁcacy in the treatment of bipolar disorder in the late 1940s by John Cade, lithium has been widely used in the treatment of acute manic symptoms and mood episode prophylaxis [21]. As mentioned, a number of double-blind studies have conﬁ rmed lithium’s efﬁcacy both in the acute treatment of mania, and for mania prophylaxis [14]. Further, multiple studies have suggested that lithium may be useful for both the acute and prophylactic treatment of depression (see [22] and [23] for meta-analysis and review). Unfortunately, many of the studies addressing monotherapy antidepressant effects had with some methodological shortcomings; in particular, low sample sizes and the use of crossover study designs. In clinical experience, lithium’s antidepressant effects as monotherapy may be modest, resulting in statistical signiﬁcance but insufﬁcient clinical antidepressant effects in many cases. However, lithium has clear utility as an adjunct antidepressant in treatment refractory patients [24]. Overall, in placebo-controlled trials, lithium has been found useful as an adjunct medication for 45% of patients, the majority of them refractory to previous treatments [25]. Lithium also has antisuicidal effects, which appear to be separate from the mood-stabilizing effects of the drug [26–28]. The molecular antecedents of this are of considerable interest. Despite these attributes lithium has a narrow therapeutic window (blood serum levels 0.6 to 1.2 mM) above which side effects are invariably intolerable. Overdose can lead to severe neurological dysfunction and in some cases death. Non-CNS side effects of lithium (not uncommonly within therapeutic levels) include tremor, polyuria, polydipsia, nausea, and weight gain. Lithium can have adverse reactions with other drug classes including diuretics, NSAIDS, and other drugs that alter kidney function.

7.3

MOLECULAR EFFECTS OF LITHIUM

Much work, crossing many decades, has attempted to focus attention on discovering lithium’s relevant therapeutic target [29–33]. The hope is that a new generation of medications can be developed that share lithium’s therapeutic target without affecting those targets responsible for the side effects and narrow therapeutic window of the drug. While development of a novel lithium mimetic will probably not cure bipolar disorder or depression, it is likely revolutionize the tolerability, as SSRIs did for the treatment of depression in the 1980s (before which time tricyclic and MOAI medications, which have more severe side effects and are dangerous in overdose, were ﬁ rst-line treatments).

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At therapeutic concentrations, lithium inhibits a small number of enzymes through competition for a magnesium-binding site (see [18] for review). Lithium inhibits a group of at least four related phosphomonoesterases, which are a group of magnesium-dependent, lithium-sensitive phosphatases that, in mammals, currently includes inositol polyphosphate 1-phosphatase (IPPase), inositol monophosphate phosphatase (IMPase), fructose 1,6-bisphosphastase (FBPase), and bisphosphate nucleotidase (BPNase) [34]. Lithium also inhibits the metabolic enzymes phosphoglucomutase (PGM) [35–38] and a kinase that functions as an intermediary in numerous intracellular signaling pathways, glycogen synthase kinase-3 (GSK-3) [39,40]. For more than a decade, the most widely accepted target for lithium has been IMPase and related enzymes [41]. IMPase is an important “recycling enzyme” in the phosphoinositide signaling pathway. This second messenger system once activated (e.g., by various muscarinic, serotonergic, or glutamatergic receptors) yields two, second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is then sequentially de-phosphorylated (“recycled”) to yield inositol (in particular a form known as myo-inositol), which can be utilized to form the starting material of the phosphoinositide signaling pathway. The enzyme that removes the last phosphate group from IP3 to yield myo-inositol is the lithium inhibitable enzyme, IMPase [42]. The ﬁ nding that lithium inhibits IMPase [43,44] led to the heurisitic inositol depletion hypothesis, which posited that lithium, via inhibition of IMPase, would decrease the availability of myoinositol, and thus ultimately the amount of PIP2 (the starting material for the signaling pathway)—thereby putting a “brake” on this pathway [45]. It was further suggested that the brain is uniquely susceptible to inositol depletion, since it has limited capacity to derive inositol [45]. IPPase—mentioned above as another lithium inhibitable enzyme—acts prior to IMPase in the inositol recycling pathway, and inhibition of IPPase is likely to increase the effects of IMPase on inositol signaling similar effects [41,42]. While lithium has been shown to decrease free inositol levels in brain sections in vitro, and in the brains of rodents treated chronically with lithium [41,46], far less consistent effects have been observed on PIP2, and those positive results that exist suggest a small effect [41,47]. Lithium has also been shown to decrease myoinositol in human subjects [48]. However, the myoinositol reduction was observed after only ﬁve days of treatment, suggesting that any decrease in PIP2 signaling occurs prior to clinically relevant effects that generally occur after longer treatment [48]. Perhaps more relevant as direct mediators of a clinical response may be preclinical evidence describing the effect of lithium on downstream targets of phosphoinositol signaling (which may be due to inhibition of IMPase/IPPase). Indeed, lithium (and valproate) has been shown in cultured cells and in the rodent brain to cause selective reductions of protein kinase C (PKC) isozymes; in the case of lithium this reduction appears to result from the depletion of inositol [49]. PKC is a major downstream target of the phosphoinositide signaling pathway.

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However, despite substantial correlations, studies have not been able to deﬁ nitively show (cause and effect) that this action of lithium has therapeutic relevance. This is in large part due to the lack of novel blood-brain barrier penetrant IMPase inhibitors. Past industry efforts have attempted to develop a brain penetrant IMPase inhibitor by taking the approach of altering the primary substrate of IMPase, inositol monophosphate [50]. Compounds with sufﬁcient inhibition were developed, but thus far these compounds have failed to advance through clinical trials because of their being highly charged [51] or extremely lipophilic [52], both of which limited brain bioavailability (see [50] for review). The published crystal structure and modeling studies of IMPase may help to develop novel inhibitors [53,54]. The selective lithium-induced inositol-dependent reduction of PKC isozymes [49] led to a “proof-of-concept” study where tamoxifen—a PKC inhibitor at high concentrations—was examined in an open label clinical trial. Initial results are promising and have led to the initiation of a larger double-blind placebocontrolled trial [55].

7.4 A NEWER TARGET OF LITHIUM: DIRECT AND INDIRECT INHIBITION OF GLYCOGEN SYNTHASE KINASE 3 In 1996 it was discovered that lithium inhibited the enzyme glycogen synthase kinase-3 (GSK-3) [39,40], a highly conserved protein serine/threonine kinase ﬁ rst characterized for its role in glycogen synthesis. These ﬁ ndings raised the possibility that GSK-3 inhibition might play a role in the treatment of bipolar disorder and depression. However, the past two years has seen the emergence of exciting new biochemical, pharmacological, genetic, and rodent behavioral studies, all of which support the hypothesis that inhibition of GSK-3 represents a therapeutically relevant target for lithium’s mood stabilizing properties. Furthermore, as we discuss, more recent preclinical evidence implicates the modulation of GSK-3 in either the direct or downstream mechanism of action of many other mood stabilizer and antidepressant medications currently in use (Table 7.1). A number of chapters of this volume discuss the various functions of GSK-3 and its role in regulating intracellular signaling pathways. To maintain focus, we do not describe these functions in complete detail here, but refer the interested reader to those chapters in this book in addition to the following comprehensive reviews for further background [56–61]. Having said that, it is of signiﬁcance to mention that GSK-3 is a ubiquitous kinase in the brain, found in both neurons and glia and localized to the cytoplasm, nucleus, and mitochondria (see [62] for review). In the brain, GSK-3 is well documented to play a critical role as a mediator of apoptosis (via PI3 kinase [56]) and Wnt pathway signaling of which βcatenin is a critical regulator [63] (Fig. 7.1). The effects of insulin on CNS neurons are not fully established; however, insulin like growth factor 1 (IGF1) appears to have many similar functions in the brain (see [58,59] and Fig. 7.1).

Treatment for depression and bipolar disorder

Treatment for depression

Treatment for bipolar disorder

Model of Electroconvulsive Therapy (ECT), the most efﬁcacious therapy for severe refractory depression; also a successful treatment for refractory mania

Antidepressants

Antipsychotics

ECS (Electroconvulsive shock)

Primary treatment for bipolar disorder Primary treatment for bipolar disorder

Involvement in Mood Disorders

Lamotrigine

Valproate

Lithium

Drug or Manipulation Direct inhibitor of GSK-3 by competition for magnesium Some studies suggest direct inhibition; many studies suggest indirect inhibition Attenuates staurosporinstimulated-caspase-3 activity in neuroblastoma cells overexpressing GSK-3 Fluoxetine, imipramine, and clorgyline increase inhibitory phosphorylation in the mouse brain Clozapine (cell cultureand rats), dopamine (mice), haloperidol, and risperidone (rats) increase inhibitory phosphorylation;haloperidol, clozapine, and riperidone and clozapine increase β-catenin and GSK-3 protein levels in rats Increases inhibitory phosphorylation of GSK-3 andβ-catenin in rats

Effects on GSK-3

Study Design

Single (p-GSK-3) and multiple (β-catenin) administrations to rats

Clozapine studies were acute to 24 hour cell culture; rodent studies were variable long-term treatments

Acute injections in mice

7 days treatment in cell culture at 0.2 and 0.3 mM

In vitro, cell culture, in vivo In vitro, cell culture, in vivo

TABLE 7.1 Diverse classes of mood stabilizers and antidepressants have effects on GSK-3

[105,107]

[109,110,156]

[99,103]

[100]

[74,81,90]

[39]

References a

132 A TARGET FOR NOVEL MOOD DISORDER TREATMENTS

Can precipitate mania in susceptible individuals; used as an lithium responsive rodent model of mania

Shown in a preliminary double-blind study to be an effective adjunct treatment for depression [144]; antidepressant-like effect in the forced swim test and olfactory bulbectomy rodent models

Amphetamine

Zinc

Source: Adapted from [155]. a See text for discussion and complete references.

Times of estrogen ﬂuxation are associated with the onset of mood disorders; regulation of estrogen is sometimes therapeutic

Estrogen

Increases inhibitory phosphorylation of GSK-3 in the brain after 15 minutes; decreases inhibitory phosphorylation after 90 minutes; acute behavioral effects prevented by GSK-3 inhibition Direct inhibitor of GSK-3 by competition for magnesium

Increases inhibitory phosphorylation of GSK-3 in rats; estrogen receptor α interacts with β-catenin; GSK-3 phosphorylates ERα

In vitro and cell culture

1 hour following estrogen administration to ovariectomized rats; interaction studies were performed in cell culture, rat brain, and Drosophila Studies were performed in mice and rats

[67]

[135,157] [158]

[116,117] [119]

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133

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A TARGET FOR NOVEL MOOD DISORDER TREATMENTS

Klein and Melton’s discovery in 1996 of lithium’s direct effects on GSK-3 was later identiﬁed to be through competition for magnesium [64,65]. Lithium inhibits both GSK-3α and GSK-3β [64]). Despite negligible inhibition by other group I metal ions, the group II ion beryllium inhibits GSK-3 in both a magnesium and ATP competitive manner [39,66]. Zinc also directly inhibits GSK-3β at physiological concentrations in vitro [67]. Similar to lithium, the direct action of zinc appears to be via competition for magnesium binding (personal communication; Hagit Eldar-Finkelman, February 2005). It was originally reported that lithium exerted approximately 50% inhibition on GSK-3 at 1 to 2 mM, which would suggest 50% inhibition (or less) at therapeutically relevant concentrations of 0.6 to 1.2 mM [39,40]. However, these in vitro studies were conducted using magnesium concentrations 10 to 20 times what is believed to be found in the brain [68–72]. Since lithium’s inhibition of GSK-3 is by competition for magnesium, these data may not be reﬂective of actual in vivo potency. Furthermore in vivo inhibition of GSK-3 appears to result in increased inhibitory serine phosphorylation through a “feedback mechanism” both in cell culture [73] and in the mammalian brain [74,75]. Possible mechanisms responsible for this feedback mechanism include increased Akt activity [73], decreased activity of protein phosphatase I [76], or increased activity of PKC [77]. Thus, even if direct inhibition of GSK-3 by lithium is modest at therapeutic concentrations, in vivo mechanism(s) exist

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Figure 7.1 Glycogen synthase kinase 3 and intracellular signaling. GSK-3 regulates diverse signaling pathways in the cell. These include insulin/insulin like growth factor (IGF-1) signaling, neurotrophic factor signaling, and Wnt signaling. Insulin signaling through its tyrosine receptor kinase (Trk) receptor activates PI 3 kinase mediated signaling, resulting in inhibition of GSK-3. Inhibition of GSK-3 activates glycogen synthase and eukaryotic initiation factor 2B (eIF2B) while inhibiting insulin receptor substrate-1 (IRS-1; an inhibitor of the insulin receptor). Insulin is generally though to have minimal effects on CNS neurons; however, IGF-1 interacting with its cognate receptor appears to have similar functions. Neurotrophins (NT) act through tyrosine receptor kinase (Trk) receptors A, B, and C to activate PI 3 kinase, AKT, and inhibit GSK-3. Many effectors have been implicated in GSK-3’s neurotrophic effects, including transcription factors (such as heat shock factor-1 (HSF-1), C-Jun, and cyclic AMP response element binding protein (CREB), and recently the proapoptotic bcl-2 family member BAX. In the Wnt signaling pathway, secreted Wnt glycoproteins interact with the Frizzled family of receptors and, through Disheveled-mediated signaling, inhibit GSK-3. Stability of this process requires the scaffolding proteins AXIN and adenomatous polyposis coli (APC). Normally active GSK-3 phosphorylates β-catenin leading to its ubquitin-dependent degradation. However, when GSK-3 is inhibited in the Wnt pathway, β-catenin is not degraded, allowing for its interaction with (T-cellspeciﬁc transcription factor) TCF to act as a transcription factor. β-catenin activity is modulated by the intracellular estrogen receptor (ER), which also effects transcription of an independent set of genes. As shown in the ﬁgure, and described in the text, medications useful for the treatment of mood disorders have both direct and indirect effects on GSK-3, and GSK-3 regulated cell signaling pathways. This includes direct effects of lithium and indirect effects of antipsychotics, amphetamine, and selective serotonin re-uptake inhibitors (SSRIs). These distinct pathways have convergent affects on cellular processes such as bioenergetics (energy metabolism), neuroplasticity, neurogenesis, resilience, and survival. Thus our hypothesis is that lithium (and other medications) may act by enhancing these processes through inhibition of GSK3. However, as detailed in the text, GSK-3 modulates a number of signaling pathways not shown in the ﬁgure. It remains to be determined which pathway(s) are most relevant to lithium’s actions in the treatment of bipolar disorder and/or depression. Gi refers to Gi /G 0 ; Gq refers to Gq /G 11. See color plates.

that indirectly amplify inhibition [75]. This premise that lithium inhibits GSK-3 at therapeutic concentrations in mammals is supported by rodent studies. Lithium was ﬁ rst shown to cross the mammalian blood-brain barrier following I.P. injections in 7-day-old rats [78]. This has been followed by evidence that therapeutic concentrations of lithium (where rodents had lithium blood levels similar to those observed in patients) result in biologically relevant GSK-3 inhibition in the mammalian brain (Table 7.2). One marker, which has been utilized to study the effects of lithium in vivo, is β-catenin (Table 7.2). We, and others, have shown that lithium administration to rodents in clinically relevant paradigms (where therapeutic blood levels were achieved) increases β-catenin in the brain [79–82].

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TABLE 7.2 Evidence for lithium inhibition of brain GSK-3 at therapeutic doses Method of Administration 4 weeks in mouse chow

Gastric gavage for 3 weeks

4 weeks of once daily IP injections 4 weeks in mouse chow

9 days of lithium in rat chow 7 days in mouse chow

Single oral gavage or 7 months in mouse chow

30 days of lithium in rat chow

Description

Reference

Increased GSK-3α/β serine 21/9 phosphorylation in the cortex, hippocampus, and striatum Decreased A-beta levels in APP-Swedish transgenic mice Increased β-catenin surrounding intracranial injection site Decreased Tau aggregation in mutant Tau transgenic mice Increased β-catenin levels in frontal cortex Increased β-catenin in hypothalmus; increased TCF/LEF-driven gene expression in amygdala and hippocampus Decreased A-beta production in PDAPP mice; decreased A-beta production and plaques in GSK-3 trangenic mice Increased β-catenin levels in frontal cortex

[74,99]

[159]

[79]

[160]

[81] [80]

[161]

[82]

Source: Adapted from [155]. Note: Only those studies where blood lithium levels were documented to be within the human therapeutic range (serum levels 0.6 to 1.2 mM) are included.

7.5 EFFECTS OF OTHER THYMOLEPTIC MEDICATIONS ON GSK-3 MEDIATED SIGNALING 7.5.1

Valproate

Valproate (known as valproic acid in the uncarboxylated form), a shortchained fatty acid, had been used as an anticonvulsant in Europe for a decade before FDA approval in 1978 for the treatment of epilepsy in the United States. Interest in the potential efﬁcacy of valproate for the treatment of bipolar disorder arose out of the suggestion that facilitating the activity of an inhibitory neurotransmitter like GABA may have antimanic effects. Early

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reports of valproate utility in acute mania [83,84] were followed by deﬁ nitive controlled studies [85–87]. Valproate has proved efﬁcacy in the acute manic phases of the illness [85,86], and may be useful as prophylaxis for future manic episodes [88,89]. It has been uncertain how valproate exerts its antimanic effects. Past hypotheses have centered on its antiepileptic mechanims of action (generally believed to be through increasing levels of GABA synthesis or inhibiting sodium channel at high time of frequencies activation). However, relatively recent literature has shown that valproate has regulatory effects on GSK-3. However, both direct and indirect effects may relevant. Chen and colleagues originally reported that valproate inhibited GSK-3α and β in vitro [90]. This ﬁ nding has also been replicated by two independent groups [91–93]. Conversely, other groups have not been able to replicate this in vitro ﬁ nding [94–97]. Hall and colleagues have found signiﬁcant inhibition, but only at concentrations above therapeutic levels [98]. It is noteworthy that Werstuck and colleagues identiﬁed derivatives of valproate that were even more potent inhibitors of GSK-3, suggesting that valproate metabolites may potently interact with GSK-3 in vivo [92]. However, it should also be noted that another group was unable to ﬁ nd direct inhibitory effects of valproate metabolites [96]. It is possible that these contradictory studies of the effects of valproate on GSK-3 may be due to minor differences in substrates or assay conditions. Deﬁ nitive studies to examine the direct effects of valproate on GSK-3 are clearly warranted. While the direct effects of valproate on GSK-3 are a matter of debate, valproate exerts effects consistent with inhibition of GSK-3 in cell culture and animal models. Valproate treatment of SH-SY5Y cells at concentrations relevant to treatment results in activation of Akt and an increase in GSK-3 inhibitory phosphorylation [74]. Valproate results in a decrease in phosphorylation of MAP1B in developing neurons, an effect that parallels GSK-3 inhibitory phosphorylation [98]. Valproate is also an activator of the Wnt signaling pathway, as judged by its effect of increasing β-catenin in cell culture [90,94] and in the rat brain following 9 days of administration [81]. Acute valproate administration to mice prevents hypoxia induced decreases in phosphorylation of GSK-3 [99]. Relatedly, Li and colleagues found that another moodstabilizing anticonvulsant, lamotrigine, similar to lithium and valproate, attenuates staurosporine and heat-shock-induced caspase-3 activity in a cell line overexpressing GSK-3β [100]. Thus GSK-3 clearly has inhibitory effects on GSK-3 regulated pathways, but it is debatable whether this is direct, indirect, or both direct and indirect. 7.5.2

Antidepressants

An analysis of the ability of the serotonergic system to regulate GSK-3 was reported by Li and colleagues [101]. This group utilized a number of different

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mechanisms (including the administration of common antidepressant medications that modulate synaptic serotonin levels), and western blot analysis, to show that acute increases in serotonin modulate GSK-3β in the mouse brain. Administration of the serotonin release stimulator and re-uptake inhibitor, d-fenﬂuramine increased GSK-3 serine 9 phosphorylation in the prefrontal cortex, hippocampus, and striatum. The monamine oxidase inhibitor clorgyline had no affect when administered on its own, but enhanced GSK-3β phosphorylation when administered prior to d-fenﬂuramine. Similar results were observed with both ﬂuoxetine and imipramine in the prefrontal cortex [101]. These imipramine western blot ﬁ ndings were recently conﬁ rmed by immunohistochemistry and extended to show that administration of imipramine to mice prevents hypoxia-induced decreases in GSK-3 phosphorylation [99].

7.5.3

Electroconvulsive Seizures

Electroconvulsive therapy (ECT) is generally considered the most effective treatment for depression, and although often not as well appreciated, ECT is an efﬁcacious treatment for mania as well [102]. The requirement for anesthesia, repeated visits to the clinic, and public stigma generally limit its use to refractory cases (or situations where pharmacological antidepressants are contraindicated such as pregnancy). Electroconvulsive seizure (ECS), an animal model of ECT, alters phosphorylation of GSK-3. Roh and colleagues found that a single administration of ECS to rats resulted in an increase in brain GSK-3β phosphorylation after 10 minutes [103]. In a follow-up study, this group provided evidence that this phosphorylation may occur in an Akt-dependent manner [104]. Madsen and colleagues studied the effects, in rats, of once daily ECS on β-catenin expression [105]. They found that 10 days of ECS increased β-catenin levels in the subgranular zone of the hippocampal dentate gyrus, and that β-catenin strongly colocalized with BrdU (used as a marker of cell proliferation). These data led the authors to postulate that the increase in β-catenin may be responsible for ECS induced increased neurogenesis [105,106]. They also found that ECS increased the levels of Wnt2, a ligand that activates β-catenin-dependent Wnt signaling through inhibition of GSK-3 [105].

7.5.4

Antipsychotics

The effect of clozapine on GSK-3 mediated signaling pathways has been examined in cell culture. Kang and colleagues found that similar to antidepressants, ECS, lithium, and valproate, clozapine administration increases inhibitory (serine 9) phosphorylation of GSK-3β [107]. This ﬁnding was coin-

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cident with an increase in nuclear β-catenin levels. While this study did not address the effects of clozapine in vivo, chronic treatment of mice with haloperidol increased serine 9 phosphorylation of GSK-3β [108]. The authors postulated that this was via an AKT-dependent mechanism, as the same treatment increased AKT phosphorylation. Chronic treatment may be required as a study by another group found that a single injection of haloperidol to mice did not increase phosphorylation of GSK-3β in the frontal cortex one hour following administration [101]. More recently Alimohamad and colleagues found that administration of haloperidol, risperidone, or clozapine increased levels of β-catenin, GSK-3 total protein, and GSK-3β serine 9 phosphorylation in regions of the rat brain [109]. They additionally examined the effects of a single injection, as well as 28 daily injections of haloperidol and risperidone, ﬁnding that only the chronic administration paradigm resulted in increases [109]. The initial western blot ﬁ ndings were conﬁ rmed with immunohistochemistry while additionally identifying increased nuclear β-catenin immunoreactivity. The protein increases were also found to occur following administration of the D2/D3 receptor antagonist raclopride. Coupled with their previous results, this ﬁ nding suggests that the mechanism of action involves inhibition of the D2 receptor [109].

7.5.5

Estrogen

A great deal of evidence suggests that gonadal steroids, and in particular, estrogen, may play a role in the pathophysiology and/or treatment of mood disorders. For example, while major depressive disorder affects 10% of men, it affects up to 2 to 3 times as many women, a ﬁgure that is only accurate following the onset of puberty. Times of estrogen hormone ﬂuctuation, such as during the postpartum and premenstrual periods, are associated with mood changes (mania and depression) [110,111]. However, little evidence suggests that absolute hormonal levels of progesterone and estrogen are different from controls [111]. This supports a notion that downstream effects of these hormones, such as receptor desensitization or abnormal signaling cascades, are responsible for the abnormal mood states. Furthermore recent studies have described the role of gonadal steroids (especially estrogen) in neuroprotection, cellular resilience, and neural plasticity—effects similar to those observed with mood stabilizers [110,112,113]. In this regard it is noteworthy that estrogen has been shown to regulate GSK-3. Estrogen administered to adult ovariectomized rats increased hippocampal serine GSK-3 phosphorylation [114]. This group further reported that the effects occurred in primary hippocampal cultures, and that GSK-3 was co-localized in a complex that included estrogen receptor α (ERα) and βcatenin. Kouzmenko and colleagues similarly immunoprecipated ERα and β-catenin, ﬁ nding that this interaction was enhanced by the addition of

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estrogen [115]. They further identiﬁed that that ERα and β-catenin could act in concert to drive gene expression by using both TCF/LEF binding element (TBE; topﬂash construct) and estrogen response element (ERE) reporting vectors. Using the chromatin immunoprecipitation (ChIP) assay, they also identiﬁed interactions of ERα and β-catenin antibodies with genes containing TBE and ERE, respectively. These data are consistent with a recent ﬁ nding, albeit in nonneuronal tissue, that concomitant lithium and estrogen administration to ovariectomized mice results in an increase in estradiol-induced proliferative and morphogenetic changes in the uterus [116]. A recent study has also identiﬁed ERα as a phosphorylation target of GSK3 [117]. This report suggests that GSK-3’s actions on ERα modulate transcriptional activity upon ligand binding, and stabilizes ERα under resting conditions.

7.6 SUPPORT FROM STUDIES OF HUMAN GENETICS AND RODENT BEHAVIOR 7.6.1

Genetics

In interpreting this section, it is important to keep in mind the general caveats of genetic association studies, which are fraught with poor reproducibility due to population stratiﬁcation artifacts. However, recent genetic evidence are supportive of a role of GSK-3 in the pathophysiology of the disorder. GSK-3α and β are mapped to human 19q13.1-13.2 and 3q13.3-21.1, respectively [118,119]. Bipolar disorder linkage studies have suggested some degree of linkage to both regions [120], though these ﬁ ndings do not approach criteria for genome-wide signiﬁcance [121]. A common GSK-3β promoter polymorphisms, (−50 T/C; [122]), has been studied in patients with bipolar disorder. While an initial family-based association study (transmission disequilibrium test) was negative [123], evidence suggests that GSK-3 polymorphisms may be associated more strongly with bipolar disorder endophenotypes [12,124,125]. Benedetti and colleagues reported that this polymorphism is associated with response to lithium in a group of 88 bipolar disorder type I patients [126]. Speciﬁcally the carriers of the T/T genotype improved less on lithium than either the T/C or C/C groups. This group has additionally studied other factors that may be associated with the −50T/C polymorphism. In a sample of 185 Italian bipolar patients, they identiﬁed an earlier age of onset associated with the T genotype (P = 0.0047) [127]. A follow-up study of another group of patients conﬁ rmed the age of onset effect of the T genotype [125]. They further identiﬁed a second association with an antidepressant response to sleep deprivation. Sixty bipolar depressed patients were administered a night of total sleep deprivation followed by a night of undisturbed sleep. Patients with the C/C genotype had better acute response to total sleep deprivation, and a subsequent higher relapse following one night of recovery sleep.

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141

Overall, these ﬁ ndings led to speculation that the C/C genotype may characterize a milder form of the disorder, with later age of onset and a better response to treatment [125]. However, it should be kept in mind that the associations to response to lithium and sleep deprivation both require replication in a distinct patient group. Further, while Benedetti and colleagues did reproduce their age of onset ﬁ ndings in a separate sample, it is likely that the second sample was derived from a similar population.

7.6.2

Rodent Behavioral Models

While there exists a lack of lack of solid animal models for most psychiatric disorders that can be utilized for in-depth molecular, biochemical, and histological analysis, some models are reasonably sound in modeling face validity, predictive validity, and predictive validity. These models attempt to recapitulate certain quantiﬁable facets of the disorders (referred to as endophenotypes [124]), predict the response of future medications based on the effects of existing drugs, or modulate causes of the underlying disease pathology itself. Since the true pathophysiology of mood disorders is unknown, and work on susceptibility genes is still in progress, the former two approaches have proved most successful. Recent evidence suggests that many of the behavioral effects of lithium in rodent models are due to inhibition of GSK-3. Dopamine systems play critical roles in regulating motoric activity and also motivational and reward circuits. Loss of motivation is one of the central features of depression and anhedonia/extreme hedonia are among the most deﬁ ning characteristics of bipolar depression and mania, respectively. Further the changes in activity present in bipolar disorder are additional characteristics of the illness, ranging from robust immobility to the profound hyperactivity of manic states. Lithium attenuates amphetamine hyperactivity in rodents, and represents a well-studied and reproducible models of antimanic drug efﬁcacy (see [128] for discussion). The hyperactivity is attenuated by antipsychotic drugs as well as anticonvulsants often utilized for the treatment of bipolar disorder [129]. It additionally has face validity; amphetamine commonly precipitates manic episodes in susceptible individual who suffer from bipolar disorder, and some studies suggest the effect is attenuated by lithium [130–132]. Beaulieu and colleagues have recently found in mice that amphetamineinduced increases in activity is mediated, at least in part, through a GSK-3 dependent mechanism [133]. This group reported that amphetamine administration to mice resulted in a reduction in the phosphorylation of GSK-3α and GSK-3β in the striatum, an effect opposite to lithium administration. They additionally reported that the endogenous hyperactivity found in dopamine transporter (DAT) knockout mice was attenuated by administration of lithium, in addition to ﬁve other GSK-3 inhibitors. Furthermore, in mice

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lacking one copy of GSK-3β, amphetamine hyperactivity was decreased. Pharmacological inhibition of GSK-3 also attenuates amphetamine hyperactivity in rats [134]. O’Brien and colleagues identiﬁed another lithium dependent behavior in mice; they found that lithium treatment results in an antidepressant-like effect (increased activity) in the forced swim test (FST) [80]. Hagit EldarFinkelman and colleagues have found similar results following intraventricular injection of lithium to mice (unpublished personal communication, February 2005). The FST developed by Porsolt and colleagues is widely utilized as a behavioral measure of antidepressant efﬁcacy [135]. The speciﬁcity of the effect of lithium is supported by studies utilizing alternate GSK-3 inhibitors. In particular, Kaidanovich-Beilin and colleagues reported antidepressant-like effects in the FST paradigm following intracerebral ventricle injections in mice of L803-mts (a peptide substrate-competitive inhibitor) [138]. This effect is concomitant with an increase in cytosolic β-catenin [136]. Gould and colleagues similarly found antidepressant-like effects in the FST following peripheral administration of AR-A014418, a selective ATP binding site inhibitor, to rats [134]—a ﬁnding that was coincident with an increase in cytosolic β-catenin [82]. Furthermore O’Brien and colleagues examined the behavioral effects of knocking out a single copy of GSK-3β, observing in these animals the same antidepressant-like behavior in the FST induced by pharmacological inhibition of GSK-3 [80]. Supportive evidence comes from behavioral studies of zinc, which is an inhibitor of GSK-3β [67]. Zinc has antidepressant-like properties in the forced swim test and olfactory bulbectomy model in rats [137–139], and sub-effective doses of zinc combined with sub-effective doses of antidepressants have adjunct antidepressant-like effects in preclinical models [137,140,141]. Furthermore, in a recent placebo-controlled clinical study, adjunctive treatment with zinc had antidepressant effects [142]. These data suggest the possibility that zinc may exert these effects through inhibition of GSK-3; however, zinc has other biological effects, including antagonism of NMDA receptors (see [19,143] for a review), that require consideration. It is quite interesting that prototypical models of depression (FST) and mania (attenuation of amphetamine hyperactivity) are modulated by GSK-3.

7.7

CONCLUDING REMARKS

The discussion above describes preclinical evidence implicating the modulation of GSK-3 mediated signaling pathways in the mechanism of action of medications used for the treatment of mood disorders. Speciﬁcally, GSK-3 is a direct target of lithium, and as was discussed, studies with rodent models suggest that both the antidepressant and antimanic actions of this medication may be mediated through inhibition of this enzyme. Further, evidence sug-

CONCLUDING REMARKS

143

gests that GSK-3 is regulated by many other medications used for the treatment of mood disorders (Table 7.1). Furthermore, although not discussed extensively in this review, numerous recent studies have provided a more complete understanding of GSK-3’s role in diverse neurological processes strengthening the hypothesis that GSK-3 may represent a therapeutically relevant target of lithium. These include neuroprotective effects, modulation of circadian rhythms, modulation of monaminergic mediated signaling events, response element binding of neurohormones and βcatenin, and metabolic effects [144]. These distinct pathways have convergent effects on cellular processes such as bioenergetics (energy metabolism), neuroplasticity, neurogenesis, resilience, and survival (see [144] for a current review). Thus our hypothesis is that lithium (and other medications) may act by enhancing these processes through inhibition of GSK-3. As discussed extensively in other chapters of this book, there exists major pharmaceutical interest in the development of novel, potent inhibitors of GSK-3, and many companies have a GSK-3 inhibitor development program (see [20,60,145–147] for more detailed discussions). The majority of current efforts have resulted in small molecule inhibitors that act by inhibiting the interaction of GSK-3 with ATP. Many of these inhibitors have had some effects on other kinases, most commonly cyclin-dependent kinases (CDKs). Thus much current effort is being placed on designing/testing drugs with greater speciﬁcity [148]. One approach has been to develop inhibitors that are non-ATP competitive [149]. For example, Eldar-Finkelman and colleagues have reported the development of L803-mts, a substrate competitive inhibitor that is active in rodent models of both diabetes and depression [136,150]. Competition for substrate, and through other non-ATP competitive approaches, has the potential to inhibit GSK-3 action in some pathways but not others [151]. These drug development efforts will hopefully allow the testing of the hypothesis that lithium, and perhaps other thymoleptic medications, exerts its therapeutic actions in the treatment of mood disorders through inhibition of GSK-3 [151]. For far too long, the accepted outcome of treatment for mood disorder has been incomplete improvement in some patients who are responsive to the current pharmacopeia. As clinical researchers, we cannot continue to be accepting of the status quo. The ultimate goal must be to strive for full remission in all patients. To reach this goal, truly novel medications must be developed. Advances in understanding targets of current medications will allow the ability to test novel compounds for efﬁcacy. It is likely that future strategies will account for disease heterogeneity subclassifying, and subsequently treating mood disorders based on endophenotypes [12,124,152]. This approach to medication development should ultimately yield truly novel and speciﬁc medications for the treatment of severe mood disorders. These medications will more speciﬁcally target the underlying pathophysiology(s) of the disorder, thus providing targeted treatment for all patients, with all forms of these heterogeneous illnesses.

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ACKNOWLEDGMENTS Research supported by the Intramural Research Program of the National Institute of Mental Heath, the Foundation for the National Institutes of Health (Neuroscience Research Fellowship (TDG)), National Association for Research on Schizophrenia and Depression (Young Investigator Award to TDG), and Stanley Research Foundation (HKM).

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8 GSK-3 AND STEM CELLS Shuibing Chen and Sheng Ding Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA

Contents 8.1 8.2

Stem Cells Wnt Signaling and Stem Cells 8.2.1 Wnt and Hematopoietic Stem Cells 8.2.2 Wnt and Intestinal Stem Cells 8.2.3 Wnt and Epidermal Stem Cells (EPSCs) 8.2.4 Wnt and Neural Stem Cells (NSCs) 8.2.5 Wnt and Neural Crest Stem Cells 8.2.6 Wnt and Myogenic Progenitor Cells (MPCs) 8.2.7 Wnt and Mesenchymal Progenitors 8.3 GSK-3 Inhibitors and Stem Cells 8.3.1 Embryonic Stem Cell Self-Renewal 8.3.2 Neural Progenitor Cell Differentiation 8.4 Concluding Remarks

8.1

STEM CELLS

Stem cells are unspecialized cells with the unique abilities to self-renew for long periods of time and to differentiate into specialized cell types in response to appropriate signals [1]. Stem cells are traditionally categorized into two classes: embryonic stem cells (ESCs) and adult stem cells. ESCs are derived Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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from the inner cell mass (ICM) of the early embryo at the blastocyst stage. They possess an unlimited capacity of self-renewal, and the pluripotency, which is the ability to develop into any cell type in the three primary germ layers of the embryo (endoderm, mesoderm and ectoderm), as well as germ cells [2–4]. In contrast, adult stem cells, found in differentiated tissues, have limited self-renewal capacity and generally can only differentiate into specialized (mature) cell types of the tissue in which they reside (multipotency). For example, the hematopoietic stem cells (HSCs), currently the best characterized multipotent stem cell population, can give rise to all the cell types of the blood but have limited ability to generate mature cells of other tissues. Along the multi-step differentiation process, stem cells develop into more lineagerestricted progenitor or precursor cells, which further mature into terminally differentiated cell types that have characteristic morphologies and specialized functions, such as muscle cell contraction or nerve cell spiking [1]. Recent advances in stem cell biology may provide new approaches for the treatment of a number of diseases and tissue/organ injuries, including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes, and spinal-cord injuries [1]. These approaches could involve cell replacement therapy and/or drug treatment to stimulate the body’s own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous stem/progenitor cells. In addition there is a growing body of evidence supporting the notion that tumors are initiated and maintained by a small number of cancer cells with stem cell related features: normal and cancer stem cells share similar self-renewal mechanisms; deregulation of signaling pathways involved in stem cell selfrenewal is associated with oncogenesis; cancer stem cells may arise from normal stem cells or through transformations of progenitor cells [5]. A better understanding of stem cell biology may also contribute to development of improved therapeutics for cancers. Stem cell fate is determined by both intrinsic regulators and the extracellular environment (niche), and is typically manipulated by “cocktails” of growth factors, signaling molecules, and/or genetic methods. During stem cell development, a precise balance is controlled among different cellular events including self-renewal, differentiation, and apoptosis (cell death). Self-renewal is the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter stem cells without the loss of developmental potential. The ability of stem cells to self-renew is critical to the development and maintenance of adult tissues. Differentiation is a process involving unspecialized cells progressing to become specialized cells with restricted developmental potentials. Under appropriate conditions in cell culture, stem cells can differentiate spontaneously. For example, the most commonly used method for inducing differentiation of mESCs involves growing them in suspension (in the presence of serum and absence of supplemented leukemia inhibitor factor [LIF]) to form aggregates called embryoid bodies (EBs) that begin to differentiate spontaneously into various cell types,

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including hematopoietic, endothelial, neuronal, and cardiac muscle cells. However, such in vitro spontaneous differentiation of EBs involves a poorly deﬁ ned, inefﬁcient, and relatively nonselective process, and therefore leads to heterogeneous populations of differentiated and undifferentiated cells. Consequently dissecting stem cell signaling pathways and identifying critical proteins that are involved in tissue speciﬁcation are essential for developments of stem cell therapy and related small molecule therapeutics. Fundamental developmental signaling pathways (Notch, Hedgehog/Hh, bone morphogenetic protein/BMP, Wnt, etc.) that control embryonic patterning and cell behaviors play important roles in stem cell regulation. For example, Notch signaling is involved in regulating self-renewal in different types of stem cells, such as HSC, neural stem cell (NSC), and intestinal stem cells (ISC) [6]. Hh signaling has been implicated in the proliferation of NSCs [7], and osteochondrogenic differentiation of mesenchymal stem cells (MSCs) [8]. BMP signaling plays essential roles in self-renewal of mESCs, HSCs, and neural crest stem cells (NCSCs), as well as in differentiations of hESCs, MSCs, and NSCs [6,9,10]. Wnt signaling has been identiﬁed in various organisms, ranging from hydra to human, and its role in development and stem cell fate regulation has been extensively studied in a variety of experimentally tractable systems. GSK-3, as a key component in Wnt signaling, and its inhibitors have been showed to regulate self-renewal and differentiation in embryonic and adult stem/ progenitor cells.

8.2

WNT SIGNALING AND STEM CELLS

Wnt proteins are a large family of secreted lipid-modiﬁed glycoproteins that are expressed in a wide variety of tissues, and may regulate canonical or noncanonical Wnt signaling pathways by different members with distinct mechanisms and functions. Canonical Wnt signaling pathway, which is by far the best characterized one, involves β-catenin as a key component and is highly conserved in evolution. Canonical Wnt signaling is initiated by Wnt protein (e.g., Wnt1 or Wnt3a) binding to a core membrane receptor complex composed of a Frizzled (Fz) receptor and a member of low-density lipoprotein (LDL) receptor-related protein (LRP5/LRP6). This binding leads to activation of a cytoplasmic protein Disheveled (Dsh), which in turn inhibits the activity of a multiprotein complex that is involved in degrading β-catenin. The multiprotein complex minimally consists of axis inhibitor (Axin), adenomatous polyposis coli (APC), and the serine/threonine kinases: casein kinase 1 (CK1) and glycogen-synthase kinase 3β (GSK-3β). Normally in the absence of Wnt stimulation, β-catenin binds with Axin and APC, and is phosphorylated by CK1 and GSK-3β in a sequential manner on at least four conserved Nterminal serine and threonine residues. This creates a recognition motif for

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an E3-ubiquitin-ligase complex that contains β-transducin-repeat-containing protein (β-TRCP), which targets phosphorylated β-catenin for ubiquitination and proteolysis. Once the kinase activity of the Axin-APC-CK1-GSK-3β complex is inhibited by the Wnt signal, β-catenin is stabilized in the unphosphorylated form, accumulated in the cytosol, and then translocated to the nucleus where it interacts with members of the LEF1/TCF family of transcription factors and regulate expression of target genes [11]. A large number of target genes regulated by this pathway have been identiﬁed, such as Cyclin D1 and c-Myc [12]. In contrast to canonical Wnt/β-catenin signaling, the mechanisms of noncanonical Wnt signaling pathways are much less clear. Two noncanonical Wnt signaling pathways have so far been proposed, referred to as the Wnt/PCP (planar cell polarity) and Wnt/Ca2+ pathways [13]. Both pathways do not involve β-catenin. The knowledge of Wnt/PCP pathway, which involves Fz and Dsh as shared components with the canonical Wnt pathway, is largely derived from studies of PCP in Drosophila tissues. This pathway is involved in regulating cytoskeletal elements and possibly JNK. The Wnt/Ca2+ pathway also involves Fz and Dsh proteins but leads to the release of intercellular calcium, which regulates the activity of calcium-modulated kinases such as CamKII and PKC [14]. Since this ﬁeld of research on Wnt signaling pathways and stem cells is broad and the function of GSK-3 inhibitors in regulating stem cell fate is mainly mediated through canonical Wnt pathway, only selected representative examples will be highlighted in this chapter. 8.2.1

Wnt and Hematopoietic Stem Cells

HSCs are perhaps the best characterized stem cells, and have long been used for treating a variety of malignant and nonmalignant hematological diseases (e.g., cancers and autoimmune disorders). Since their identiﬁcation in 1960s, great progress has been made in understanding HSC biology, and their isolation and transplantation. In mice the long-term self-renewing HSCs make up approximately 0.007% of bone marrow and can be isolated by FACS based on their expression of high levels of c-Kit and Sca-1, low levels of Thy-1 and undetectable levels of lineage markers (e.g., CD3, Mac-1, and B220). When mHSCs are transplanted into lethally irradiated mice, these cells repopulate the bone marrow and balance self-renewal and differentiation to maintain the hematopoietic system for the life of the animals. hHSCs can be similarly identiﬁed and isolated through the expression of cell surface antigens (e.g., CD34, CD38) and the absence of lineage markers. Xenotransplantation of hHSCs into nonobese diabetic/severe combined immunodeﬁcient (NOD/ SCID) mice has proved to be a reliable model for examining the primitive long-term human blood stem cells with repopulating capacity [15]. The ability to prospectively identify and purify HSCs has also allowed recent advancement in understanding the molecular control of their activities, particularly self-renewal and maintenance. There is growing evidence

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suggesting that Wnt signaling directly promotes hematopoietic stem/progenitor cell self-renewal. Wnt3a, a soluble Wnt protein, was shown to synergize with Steel factor (SLF) to promote the proliferation and inhibit the differentiation of murine hematopoietic progenitors in vitro [6]. Treatment with Wnt3a or overexpression of activated β-catenin expanded long-term repopulating mHSCs in cell culture, while ectopic expression of Axin or a Fz ligandbinding domain, inhibitors of the Wnt signaling pathway, led to inhibition of mHSC self-renewal in vitro and reduced their in vivo reconstitution capability [16]. Furthermore activation of Wnt signaling in HSCs increased expression of HoxB4 and Notch1, which were previously implicated in promoting selfrenewal of HSCs [17]. Noncanonical Wnt signaling may also have an effect on enhancing self-renewal of hematopoietic stem/progenitor cells. Wnt5a treatment of human hematopoietic progenitors in the presence of stromal cell contact promoted the expansion of undifferentiated progenitors in vitro. In addition treatment of mice with Wnt5a-conditioned medium (CM) resulted in increased hHSC population in a NOD/SCID xenotransplant model [18]. All of these evidences suggest that Wnt signaling plays a role in regulating murine and human HSC self-renewal. In addition to its role in directly regulating HSC self-renewal, Wnt signaling could indirectly modulate HSC self-renewal by inﬂuencing the HSC niches. HSC niches are supportive microenvironments composed of a heterogeneous population of cells, including ﬁbroblasts, adipocytes, endothelial cells, and osteoblasts. Wnt signaling plays an important role in the formation of osteoblasts, which support HSCs in the niches. This is supported by the observations such as loss-of-function mutations in LRP-5 in both human and mice result in reduced bone mass, overexpression of an activated form of human LRP-5 leads to endosteal hyperostosis and increases trabecular bone volume, and increasing the number of osteoblasts in speciﬁc regions of bone causes parallel increases in the long-term HSC population. Wnt signaling can also inhibit adipogenesis of mesenchymal progenitors and preadipocytes. Because increasing the number of mature adipocytes can inhibit hematopoiesis, Wnt signaling might also contribute to the maintenance of the HSC niche by regulating adipogenesis and the number of mature adipocytes. As osteoblasts and adipocytes are derived from common mesenchymal precursors, it is also possible that Wnt signaling is involved in the development and maintenance of the HSC niches through regulating the ratio of osteoblasts to adipocytes [19]. 8.2.2

Wnt and Intestinal Stem Cells

The intestinal epithelium is a self-renewing tissue that represents a unique system for studying interconnected cell behaviors such as proliferation, differentiation, and migration. The epithelium of intestine is organized into two morphologically and functionally distinct compartments: proliferative crypt, which is maintained by multipotent stem/progenitor cells, and differentiated

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villus, the cells of which are derived from several surrounding crypts. Slowly dividing ISCs are postulated to reside at the base of each crypt. They can give rise to a population of immediate cell descendants referred to as transit amplifying (TA) cells. The TA population undergoes rapid proliferation and further expands into a population of nonproliferating daughter cells that gradually differentiate into four principal epithelial lineages: absorptive cells or enterocytes, mucus-producing goblet cells, enteroendocrine cells, and Paneth cells. The differentiation process is coupled with cell migration upward. Recently musashi-1 was identiﬁed to be quite speciﬁcally expressed in ISCs and considered as a putative marker gene for ISCs [20]. Despite difﬁculties in characterizing ISCs, several studies have suggested the Wnt/β-catenin pathway in the maintenance of ISCs. First, proliferative cells at the bottom of the colon crypts accumulate nuclear β-catenin. Second, loss-of-function mutation in Tcf4 led to the depletion of intestinal proliferative compartments in fetal mice [21,22]. Third, targeted expression of Dickkopf-1 (Dkk-1), a secreted potent Wnt antagonist, under the control of a 9-kb regulatory region of the mouse villin gene reduced the epithelial proliferation in villus with the loss of crypts [23]. Moreover treatment of adult mice with adenovirus expressing Dkk1 repressed expression of the Wnt target genes, CD44 and EphB2, within 2 days in both small intestine and colon, followed by the marked inhibition of proliferation in these regions and a progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. This degeneration is a reversible process, since decreased Dkk1 expression at late time points (>10 days) is followed by crypt and villus regeneration [24]. 8.2.3

Wnt and Epidermal Stem Cells (EPSCs)

Mammalian epidermis, which is a far more complex structure than the cryptvillus unit, consists of a multilayered epithelium, the interfollicular epidermis (IFE) with associated hair follicles (HFs), sebaceous glands (SGs), and sweat glands. Epidermal maintenance depends on stem cells. Because long-term EPSCs divide infrequently, they can be visualized as BrdU DNA labelretaining cells (LRCs). LRCs are concentrated in the bulge area and express markers such as CD34 and keratin 15 (K15) [25]. Multipotent EPSCs can generate the hair lineages, sebocytes, as well as the stem cells of the interfollicular epidermis. At the beginning of each hair cycle, EPSCs are stimulated to generate a rapidly proliferating hair germ. While the hair germ grows/migrates downward, the EPSCs reenter quiescence. The hair germ then envelopes specialized mesenchymal cells and forms a matrix of proliferative TA cells at the base of the follicle. The matrix cells terminally differentiate to produce the inner root sheath and hair shaft [26]. Studies have suggested that multiple steps in morphogenesis and cycling of the follicle are dependent on Wnt signaling. For example, gain- and loss-offunction studies have demonstrated that Wnt/β-catenin activation is essential

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in promoting the transition/conversion from quiescent EPSCs to TA cells and their subsequent proliferation to form the activated cells of the newly developing hair follicle [27]. Additional studies showed that epidermal expression of activated β-catenin led ﬁ rst to induction of hair growth and then to de novo HF formation from IFE, SG, and preexisting HF. By contrast, ablation of β-catenin prevented new HF formation and disturbed growth of existing HF. Using K14-ΔNβ-cateninER transgenic mice, in which β-catenin is controllably activated in the basal layer of the epidermis by 4-hydroxytamoxifen, it was shown that short-term, lowlevel β-catenin activation stimulated de novo hair follicle formation from SG and IFE, while only sustained, high-level β-catenin activation induces new follicles from preexisting follicles. The β-catenin-induced follicles contain keratinocytes with three characteristics of bulge stem cells: clonal growth in culture and expression of K15 and CD34. When β-catenin is activated, Hh pathway is upregulated, and inhibition of Hh signaling converts the low-level β-catenin phenotype to the wild-type epidermis and the high-level β-catenin phenotype to the low-level one. These results demonstrated the remarkable capacity of adult epidermis to be reprogrammed by titrating β-catenin [28]. 8.2.4

Wnt and Neural Stem Cells (NSCs)

During embryonic, neonatal, and adult neurogenesis, regulated proliferation and differentiation of neural stem/progenitor cells are essential for proper development and maintenance of the neural tissues in the vertebrate central nervous system (CNS). Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from the studies of mouse and chick models. In the CNS, Wnt3a is required for neural progenitor proliferation and hippocampal development, while the absence of both Wnt1 and Wnt3a affects the expansion of dorsal neural cells. Activation of Wnt signaling by ectopic expression of Wnt proteins or constitutively activated βcatenin reduces neuronal differentiation, increases progenitor numbers, and consequently leads to a massive enlargement of neural tissue in certain regions of the CNS [29]. However, it is still unclear whether this phenotype is due to effects of Wnt/β-catenin activation on multipotent, self-renewing NSCs and/ or progenitors. In the developing Xenopus retina, Fz5 and Wnt protein selectively regulate neural potential by regulating the expression of Sox2, which is a master transcription factor controlling the formation and maintenance of neural-competent precursors. Blocking either Fz5 or canonical Wnt signaling within Xenopus retina suppressed Sox2 expression, reduced cell proliferation, inhibited the onset of proneural gene expression, and biased progenitors toward a nonneural fate, without altering the expression of multiple progenitor markers [30]. Wnt signals are also shown to regulate neural differentiation at cellular level. Wnt3a promotes differentiation of embryonic NSCs that are dissociated from neurospheres derived from embryonic day (E) 11.5 mouse telencephalon.

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CM containing Wnt3a can inhibit the regeneration of neurospheres and promote NSC differentiation into MAP2-positive neuronal cells. Wnt3a CM can also increase the number of glial ﬁbrillary acidic protein (GFAP)-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4 [31]. In another study, Wnt signaling was shown to have stage-speciﬁc effects on regulating mouse neural precursor cells (NPCs) in vivo and in culture prepared from neocortex. In the early NPCs (E8.5–10.5), Wnt signaling and stabilized β-catenin seemed to reduce neuronal differentiation, while in the late NPCs (E11.5–13.5), Wnt signaling inhibited their proliferation, and instructively promoted their neuronal differentiation. Overexpression of Wnt7a or a stabilized form of β-catenin in late-stage mouse NPC cultures induced neuronal differentiation even in the presence of ﬁbroblast growth factor 2 (FGF2), which is a self-renewal promoting (proliferation) factor in this system. Moreover the β-catenin/TCF complex can directly regulate the promoter of neurogenin 1, a master gene involved in cortical neuronal differentiation [32]. 8.2.5

Wnt and Neural Crest Stem Cells

NCSCs are multipotent stem cells that give rise to an impressive array of cell types, including most structures of the peripheral neurous system (PNS) and nonneural tissues such as cells in the outﬂow tract of the heart, craniofacial bone and cartilage, connective tissues, and melanocytes of the skin. NCSCs arise within the developing CNS and subsequently migrate away, sometimes moving extremely long distances to populate peripheral region of the embryo [29]. Wnt proteins promote the induction of neural crest from neural plates, while inhibition of Wnt signaling perturbs neural crest formation in vivo. Wnt1 and Wnt3a are expressed in the dorsal part of the neural tube at the time of neural crest emigration, while the deletion of both Wnt1 and Wnt3a results in a marked reduction of neural crest derivatives [33]. Wnt/β-catenin signaling activation in emigrating NCSCs has little effect on proliferation and instead regulates fate decisions. Sustained β-catenin activity in NCSCs promotes the formation of sensory neuronal cells in vivo at the expense of virtually all other neural crest derivatives [34]. Consistently Wnt1 is able to instruct early NCSCs to adopt a sensory neuronal fate in a β-catenin-dependent manner [35]. This sensory fate-inducing activity of Wnt/β-catenin is antagonized by BMP signaling [36]. 8.2.6 Wnt and Myogenic Progenitor Cells (MPCs) Skeletal muscles contain a progenitor cell population with high expression of cell surface markers CD45 and Sca1 [37]. These CD45 + /Sca1+ muscle progenitor cells can undergo myogenic commitment during muscle regeneration, and can be readily isolated from experimentally injured muscle. Stimulation of Wnt signaling by lithium (a GSK inhibitor) in freshly isolated CD45 + /Sca1+

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MPCs is sufﬁcient to induce muscle speciﬁcation. In addition co-culture with cells ectopically expressing Wnt proteins could induce Pax7 induction and myogenic commitment of CD45 + /Sca1+ MPCs. Regenerating muscles treated with sFRPs (which are soluble Wnt antagonists) displayed a marked reduction in the number of myoblasts derived from CD45 + /Sca1+ MPCs. All these ﬁ ndings suggest that Wnt signaling is a mechanism by which CD45 + /Sca1+ MPCs are induced to undergo myogenic speciﬁcation. Interestingly sFRPs are upregulated in the late stage of muscle regeneration when myoﬁbers are well formed. This might be a feedback mechanism that late expression of sFRPs inhibits the Wnt-dependent recruitment of myogenic progenitors after regeneration is complete [38]. 8.2.7 Wnt and Mesenchymal Progenitors Involvement of canonical Wnt signaling pathway in stem cell fate regulation is also supported by studies in other tissues, such as vertebrate skeleton. Skeletal development and maintenance depend on proliferation, differentiation, and coordinated activities of chondrocytes, osteoblasts, and osteoclasts. Chondrocytes and osteoblasts are thought to differentiate from the common mesenchymal precursors, osteochondroprogenitors [39]. Recently compelling evidence suggests that Wnt signaling represents both a cell-autonomous mechanism for inducing osteoblastic, and suppressing chondrocytic differentiations in early osteochondroprogenitors, and a mechanism in fully differentiated osteoblasts for inhibition of osteoclasts formation from osteoclastic progenitors by stimulating the production of osteoprotegerin (OPG). βcatenin is required at the early stage to repress chondrocytic differentiation [40]. Conditional deletion of β-catenin in the limb and head mesenchyme during early embryonic development resulted in inhibition of osteoblastic differentiation and lack of mature osteoblasts in membranous bones. Such ablation of β-catenin induced the osteochondroprogenitors to differentiate into chondrocytes instead of osteoblasts. On the other hand, differentiated osteoblasts with high β-catenin levels have a strong inhibitory effect on osteoclast differentiation. This is based on the observation that transgenic mice, in which β-catenin is constitutively stabilized in differentiated osteoblasts, showed failure of tooth eruption, a massive increase in bone mass in vertebral and limb bones, and a decrease in the number of osteoclasts [41]. This inhibitory effect is due to TCF-1 induced upregulation of OPN, which inhibits osteoclastogenesis by competing with the receptor activator of the nuclear factor-KappaB (RANK) receptor on osteoclastic progenitors for binding to the RANK ligand. These studies may provide novel therapeutic strategies to treat skeletal diseases [42]. Wnt signaling can also inhibit adipogenesis of both mesenchymal progenitors and preadipocytes. In mesenchymal progenitors, stabilization of β-catenin promotes osteogenesis, inhibits adipogenesis, and regulates other aspects of cell fate determination [43]. In preadipocytes, 3T3L1 cells, Wnt receptors are

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expressed, and their expressions decline upon induction of adipogenic differentiation. Wnt signaling maintains preadipocytes in the undifferentiated state through inhibition of adipogenic transcription factors CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of Wnt signaling in preadipocytes by overexpression of Axin or domain-negative TCF4 induces the cells to differentiate into mature adipocytes. Disruption of Wnt signaling also causes transdifferentiation of myoblasts into adipocytes in vitro [44]. Wnt signaling pathway plays critical roles in regulating cell fate in many types of stem cells. The speciﬁc function of Wnt signaling, in either maintaining stem cell self-renewal or inducing cell lineage speciﬁcation, depends on cell-intrinsic properties and niches. Importantly, the niches of stem cells also inﬂuence how cells interpret signaling cross-talks, by which other signaling pathways regulate Wnt signaling with synergistic or antagonistic effect. How Wnt and other signaling pathways are integrated in regulating stem cell function remains to be elucidated.

8.3

GSK-3 INHIBITORS AND STEM CELLS

GSK-3 is an unusual serine/threonine kinase that is generally active under resting conditions and is primarily regulated by inactivation through various signaling pathways. The functions of the two mammalian isoforms, GSK-3α and β, have been implicated in a variety of biological processes. However, our current knowledge on the role of GSK-3 in regulating stem cell fate has been largely centered on Wnt signaling pathway, and GSK-3 inhibitors have been identiﬁed and used by this context/mechanism. 8.3.1

Embryonic Stem Cell Self-Renewal

Human and mouse ESCs (hESCs and mESCs) are pluripotent stem cells. Conventionally, they are maintained on feeder cells and/or mixtures of exogenous factors (e.g., serum). Studies have found that the self-renewal of mESCs primarily depends on two key signaling molecules: LIF/interleukin 6 (IL6) family members and BMP (Fig. 8.1). LIF acts via a membrane-bound gp130LIFR (LIF receptor) complex to activate Stat (signal transduction and activation of transcription) signaling, which promotes self-renewal of mESCs and inhibits their differentiations to mesoderm and endoderm by a Mycdependent mechanism. BMP induces expression of Id (inhibitor of differentiation) genes via Smad signaling and inhibits mESC differentiation to neuroectoderm. The combination of BMP and LIF can sustain mESCs selfrenewal in the absence of serum and feeder cells. Recently it was shown that Wnt signaling, which is active in undifferentiated mESCs and is downregulated upon differentiation, plays an important role in ESC self-renewal. 6Bromoindirubin-3′-oxime (BIO) (Fig. 8.2), a natural product derived from

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LIF

Growth Factor

BMP

Wnt

MAPK GSK3? GSK3b

Smad STAT3

T58

Ids

pT58

c-myc

c-myc u

proteosome

Self-renewal

ES cells

Neural differentiation

Non neural differentiation

Figure 8.1

The signalling network regulating mESCs self-renewal.

Br HO

H N

O

N

NH

N H

OH

O

BIO

Figure 8.2

N

HN

N

O

Br Kenpaullone

NH2

N H TWS119

The chemical structure of known GSK-3β inhibitors.

mollusk Tyrian purple, has been recently shown to maintain mESCs in the pluripotent state without LIF, feeder cells, or conditioned medium. mESCs expanded under BIO treatment can generate teratomas consisting of all three germ layer-derived tissues, including neuroepithelium (ectoderm), cartilage (mesoderm), and ciliated epithelium (endoderm). In addition BIO-treated mESCs can contribute to chimeric animals after being injected into blastocyst. BIO is proposed to function by inhibiting GSK-3β, resulting in activation of canonical Wnt signaling pathway. To conﬁ rm such mechanism, recombinant Wnt3, a protein, was shown to have a similar effect as BIO on mESC

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self-renewal [47]. One of the downstream substrates of GSK-3β in the mESC self-renewal process is suggested to be Myc, which is also regulated by Stat3 at the transcriptional level. In undifferentiated mESCs, Myc mRNAs are expressed by an elevated level and Myc proteins are stabilized in the unphosphorylated form. Following LIF withdrawal, Myc mRNA expression is downregulated and Myc proteins become phosphorylated on threonine 58 (T58) by GSK-3β, leading to their degradation. BIO seems to play a role in suppressing GSK-3β-mediated Myc-T58-phosphorylation, sustaining Myc stability, and maintaining the pluripotency of mESCs [48]. There are fundamental differences between maintenance of mESCs and hESCs. For example, activation of Stat3 signaling is not sufﬁcient to maintain hESC self-renewal, and BMP signaling works as a differentiation inducer in hESCs [49]. In the expression analysis, Wnt proteins were found in the undifferentiated hESCs; while several Wnt proteins, most notably Wnt5a and Wnt6, are expressed in the differentiated populations, such as EBs. Two Wnt antagonists, secreted Frizzled-related protein 1 (SFRP1) and SFRP2, are upregulated in the undifferentiated hESCs and two Wnt inhibitors, Axin and protein phosphatase 2A (PP2A), are downregulated in the differentiated populations. The Wnt agonists, casein kinase 1 alpha (CSNK1A), Disheveled activator of morphogenesis 1 (DAAM1) and DAAM2 are all upregulated in EBs [49]. Interestingly, BIO-mediated ESC self-renewal is also shown in hESCs. After seven days treatment with BIO, hESCs maintain the Oct3/4 expression and compact colony morphology. hESCs treated by BIO seem to preserve their pluripotentials, as they can form differentiating EBs, which express markers of ectoderm, mesoderm, and endoderm, and also differentiate into neurons by speciﬁc culture techniques. However, such self-renewal studies have only been conducted over a brief culture period [47]. Further investigation is needed to elucidate the function of Wnt signaling in hESCs. 8.3.2

Neural Progenitor Cell Differentiation

There is increasing evidence that Wnt signaling also contributes to the patterning of the vertebrate nervous systems. Anterior-posterior (A-P) patterning of the neural tube seems to require suppression of the canonical Wnt signaling pathway. Lithium chloride, a GSK-3β inhibitor, inhibits the formation of anterior structures. During last gastrulation, the forebrain is regionalized and divided dorsally into the telencephalon and eye ﬁeld, and ventrally into hypothalamus, whereas the most posterior regions give rise to the diencephalon. Wnt inhibition is required for the regionalization of forebrain. In zebraﬁsh the Mbl (masterblind) mutant, telencephalon, and the eye ﬁeld are reduced or absent, and the posterior structures are rostrally expanded. The Mbl mutant carries a point mutation in Axin1 that affects its binding to GSK3β and results in a gain of function in Wnt signaling. Accordingly inhibition of GSK-3β by lithium or overexpression of a dominant negative form of GSK3β produces the similar phenotypes as the Mbl mutant. Analyses of the Wnt1

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mutant reveal a crucial role for Wnt signaling in the formation of the midbrain-hindbrain boundary (MHB), an important organized centre that is posterior to the forebrain. Wnt1 loss of function results in progressive loss of the posterior midbrain and anterior hindbrain as a result of cell death in the midbrain and the rostral hindbrain. A speciﬁc inactivation of β-catenin in the MHB mimics the Wnt1 loss-of-function phenotype [50]. Wnt pathway has also been implicated in regulating dopamine (DA) neuron differentiation in the developing ventral mesencephalon (VM) and therefore may be intervened for the treatment of Parkinson’s disease. Two chemical inhibitors of GSK-3β, BIO and kenpaullone (Fig. 8.2), were found to enhance differentiation of the nuclear receptor-related factor 1 positive (Nurr1+) VM precursor cells into tyrosine hydroxylase positive (TH +) DA neurons by three- to ﬁvefold, but have no effect on cell survival or proliferation of the VM precursors. In addition it was conﬁ rmed that these two GSK-3β inhibitors could stabilize βcatenin and the overexpression of β-catenin in the VM precursors resulted in increased DA neuron differentiation. These data suggests that GSK-3β inhibitors may be used to improve stem/progenitor cell therapies in Parkinson’s disease [51]. TWS119 (Fig. 8.2), a synthetic disubstituted pyrrolopyrimidine, was identiﬁed from a cell-based screen as a potent inducer of neuronal differentiation in pluripotent mESCs and P19 murine embryonal carcinoma cells (mECCs). A panel of afﬁ nity matrices, prepared from representative TWS analogues, were used to pull down target proteins from P19 cell extracts. Proteins speciﬁcally bound to all positive resins derived from active molecules but not to the negative resins derived from inactive molecules were considered to be the putative targets of TWS119. Consequently GSK-3β was identiﬁed as one target of TWS119, and conﬁ rmed by biochemical and cellular assays (surface plasmon resonance, kinase inhibition assay, western blot, reporter assay, etc.). This target identiﬁcation may provide yet another link between neuronal differentiation and the Wnt signaling pathway. Additional studies also indicated that TWS119 is not entirely speciﬁc against GSK-3β. Alternatively, TWS119 might promote neuronal differentiation of mESCs via novel mechanisms other than the canonical Wnt signaling pathway. Such mechanisms might include the inhibition of one or more kinases that were not apparent in the afﬁ nity experiments (possibly due to low abundance or other factors), or other proteins involved in controlling stem cell fate [52]. Although activation of canonical Wnt signaling pathway has been implicated in regulating various stem/progenitor cell fate under different contexts, GSK-3 inhibitors have not yet been extensively used in stem cell biology largely because of their availability, potency, and speciﬁcity. For example, some GSK-3 inhibitors that were used as strong Wnt pathway activators are not entirely speciﬁc, such as BIO and TWS119, which also inhibit Abl, CDK, Src, TrkB, and Flt3, for example, with nanomolar IC50 s. In contract, some other potent GSK-3 inhibitors, such as AR-A014418, even when used at high concentration do not activate Wnt pathway strongly. Consequently a better

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understanding of their mechanism of action in cells and continued development of more speciﬁc GSK-3 inhibitors may facilitate their application in stem cell ﬁeld. 8.4

CONCLUDING REMARKS

The Wnt signaling has emerged as a critical pathway in stem cell fate regulation, including proliferation, differentiation/patterning, migration, and survival. For years to come, studying functions of Wnt signaling and its interactions with other pathways (e.g., BMP, Hh, and Notch) during tissue/ organ development, maintenance, and regeneration will be an important and exciting area in stem cell biology. It should lead to our improved control of stem cell fate and ultimately facilitate novel approaches (e.g., GSK-3 inhibitors for regulating stem cells) to harness stem cells’ immense therapeutic potentials for treating human diseases. REFERENCES 1. Department of Health and Human Services. (2001). Stem cells: Scientiﬁc progress and future research directions. http://stemcells.nih.gov/stemcell/scireport. asp and references therein. 2. Hubner K., Fuhrmann G., Christenson L., Kehler J., Reinbold R., De La Fuente R., Wood J., Strauss J., Boiani M., Scholer H. (2003). Derivation of oocytes from mouse embryonic stem cells. Science, 300, 1251–1256. 3. Toyooka Y., Tsunekawa N., Akasu R., Noce T. (2003). Embryonic stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci., 100, 11457–11462. 4. Geijsen N., Horoschak M., Kim K., Gribnau J., Eggan K., Daley G. (2004). Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature, 427, 148–154. 5. Pardal R., Clarke M., Morrison S. (2003). Applying the principles of stem-cell biology to cancer. Nat. Rev. Cancer, 3, 895–902. 6. Molofsky A., Pardal R., Morrison S. (2004). Diverse mechanisms regulate stem cell self-renewal. Curr. Opin. Cell Biol., 16, 700–707. 7. Ho K., Scott M. (2002). Sonic hedgehog in the nervous system: Functions, modiﬁcations and mechanisms. Curr. Opin. Neurobiol., 12, 57–63. 8. Wu X., Ealker J., Zhang J., Ding S., Schultz P. (2004). Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem. Biol., 11, 1229–1238. 9. Stern C. (2005). Neural induction: Old problem, new ﬁ ndings, yet more questions. Development, 132, 2007–2021. 10. Wan M., Cao X. (2005). BMP signaling in skeletal development. Biochem. Biophys. Res. Commun., 328, 651–657. 11. Reya T., Clevers H. (2005). Wnt signaling in stem cells and cancer. Nature, 434, 843–850.

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9 GLYCOGEN SYNTHASE KINASE 3: ROLE IN NEURODEGENERATION AND NEUROPROTECTION Laura Facci and Stephen D. Skaper Neurology and GI Centre of Excellence for Drug Discovery GlaxoSmithKline Research and Development Limited Harlow, Essex, UK

Contents 9.1 Introduction 9.2 GSK-3 and Neurodegeneration 9.2.1 Alzheimer’s Disease and Tauopathies 9.2.2 Other Neurodegenerative Disorders 9.3 GSK-3 Inhibitors and Neuroprotection 9.3.1 Lithium 9.3.2 Small-Molecule Inhibitors 9.4 Concluding Remarks

9.1 INTRODUCTION Differentiated cells, including neurons in the central nervous system (CNS), require the presence of survival factors to suppress the intrinsic cell death machinery and thereby avoid apoptosis [1]. The regulation of apoptosis by survival factors is critical for normal development and proper functioning of multicellular organisms. Aberrant cell death within the adult CNS is a key Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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mechanism thought to underlie the pathology of several neurodegenerative diseases [1,2]. Survival growth factors protect neurons from a variety of proapoptotic stimuli [3,4], and one of the protective mechanisms has been attributed to the activation of the phosphatidylinositol 3-kinase (PI 3-kinase) signal transduction pathway [5–7]. A downstream effector of this signaling pathway is Akt (protein kinase B, PKB), a kinase that phosphorylates the serine/ threonine kinase glycogen synthase kinase-3β (GSK-3β; also known as tau protein kinase I) on ser9 to render it inactive [8–10], a proposed mechanism by which neurons become resistant to apoptotic stimuli [11–13]. There are two highly homologous forms of GSK-3, GSK-3α, and GSK-3β [14]. GSK-3β may also be regulated by other PI 3-kinase-dependent, but Akt-independent, pathways [15,16]. Recombinant overexpression studies using active or dominant-negative proteins have led to the proposal that GSK-3 may be a relevant effector of PI 3-kinase-mediated neuronal cell survival [11,12,17]. GSK-3β has been implicated in many cellular functions, such as the cell cycle, gene transcription, cytoskeletal integrity, and apoptosis, as a result of its ability to phosphorylate key proteins that modulate these processes [18]. This constitutive kinase is ubiquitously expressed, including abundant expression in the brain [14]. This chapter focuses on the implications of GSK-3 activation in the context of neurodegenerative disease, as well as a target for neuroprotection.

9.2 GSK-3 AND NEURODEGENERATION 9.2.1 Alzheimer’s Disease and Tauopathies Most neurodegenerative diseases are characterized by the progressive loss of neurons in speciﬁc brain regions. This is particularly true of Alzheimer’s disease (AD), the most frequent cause of dementia [19]. Senile or amyloid plaques, disturbed synaptic form, extensive neuronal loss, and neuroﬁbrillary tangles (NFTs) represent the main histological hallmarks observed in AD brains. A major constituent of amyloid plaques is the 39–43 amino acid amyloid-β-protein (Aβ), which is constitutively produced by normal cleavage of the membrane-bound amyloid-β precursor protein. Amyloid-β-containing extracellular plaques are an early and invariant feature of the disease. All known familial AD mutations lead to increased production or cerebral deposition of Aβ [20]. Abnormal Tau hyperphosphorylation and deposition in the cytoplasm of neurons and glial cells is another major structural and biochemical characteristic in AD, Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration, which constitute the group of neurodegenerative tauopathies [21]. Glycogen synthase kinase has emerged as a key kinase that is able to interact with many of the proteins implicated in the etiology of AD, as well as other tauopathies. GSK-3β accumulates in the cytoplasm of AD pretangle

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neurons and in Tau deposits in most tauopathies [22], and its distribution in AD brains is coincident with the sequence of development of neuroﬁbrillary changes [23,24]. GSK-3α has been found to co-immunoprecipitate with paired helical ﬁ laments-Tau from AD brain [25] and to co-localize with granulovacuolar degeneration in AD brains [26]. In addition GSK-3β phosphorylates those amino acid residues in Tau known to be hyperphosphorylated in paired helical ﬁ laments in transfected cells [27]. Overexpression of GSK-3β decreases the levels of β-catenin, and results in Tau hyperphosphorylation and somatodendritic localization of Tau and neurodegeneration in GSK-3β conditional transgenic mice [28]. These mice develop pretangle structures in the hippocampus, as well as increased neuronal cell death, gliosis, and spatial learning deﬁcits [29]. Overexpression of human Tau, in combination with its phosphorylation by the Drosophila GSK-3β homologue Shaggy, exacerbates neurodegeneration induced by Tau overexpression alone and gives rise to NFT pathology in the ﬂy [30]. Likewise overactivation of GSK-3 by inhibition of PI 3-kinase and protein kinase C leads to hyperphosphorylation of Tau and impairment of spatial memory in rats [31]. The recent ﬁ nding that GSK-3 is able to modulate Tau-mRNA spicing raises the possibility that GSK-3 could contribute to tauopathies via aberrant Tau splicing, as well [32]. GSK-3 has also been linked to the downstream effects of Aβ. Exposure of CNS primary neuronal cell cultures to Aβ or C-terminal fragments of amyloid precursor protein is reported to induce the activation of GSK-3β [33–35] and cell death [34–37]. Blockade of GSK-3β expression by antisense oligonucleotides [35] or its activity by lithium [38] inhibits Aβ-induced neurodegeneration of cortical and hippocampal primary neurons in culture. Activation of GSK-3β by Aβ enhances phosphorylation of Tau in hippocampal neurons [39]. Active GSK-3β triggers signal transduction events that participate in cell death [12,13,40], and loss of neuronal and synaptic plasticity [41]. Taken together, the studies above provide strong evidence that increased GSK-3β expression and activity play a signiﬁcant role in the neurodegenerative pathology that is associated with AD. GSK-3β phosphorylates and regulates the functions of many metabolic, signalling, and structural proteins. Notable among the signaling proteins regulated by GSK-3β are the many transcription factors, including activator protein-1, cyclic AMP response element binding protein, heat shock factor-1, nuclear factor of activated T cells, Myc, β-catenin, CCAAT/enhancer binding protein, and NF-κB. A discussion of these elements is beyond the scope of this chapter, and can be found reviewed elsewhere [42]. 9.2.2 Other Neurodegenerative Disorders Reports documenting GSK-3 activation in other neurological disorders are not common. Far more numerous are studies that have relied on lithium as a neuroprotective agent, to demonstrate a putative role for GSK-3 role in disease. The latter ﬁndings will be discussed in the last section.

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Prion diseases are characterized by neuronal cell death, glial proliferation, and deposition of prion peptide aggregates. An abnormal misfolded isoform of the prion protein (PrP) is considered to be responsible for this neurodegeneration [43]. The PrP 106–126, a synthetic peptide obtained from the amyloidogenic region of the PrP retains the ability to trigger cell death in neuronal cultures [44]. Addition of this prion peptide to cultured CNS neurons increases the activity of GSK-3, which is accompanied by the enhanced phosphorylation of some microtubule-associated proteins including Tau [45]. Overexpression of a dominant negative GSK-3 prevented prion peptideinduced neuronal cell death [45]. These results are consistent with the view that the activation of GSK-3 is a crucial mediator of prion peptide-induced neurodegeneration. Human immunodeﬁciency virus type 1 (HIV-1) invades the CNS and frequently causes progressive neurologic disease. One of the most salient neuropathological features of HIV-1 infection in cerebral cortex and basal ganglia is neuronal apoptosis. Neuronal apoptosis is believed to occur as a result of the production and release of a number of neurotoxic factors, which include the HIV-1 regulatory protein, Tat [46]. Addition of Tat to cultured CNS neurons resulted in an increase in GSK-3β activity [47]. The activation of platelet activating factor receptors is critical for the induction of neuronal cell death by several candidate HIV-1 neurotoxins, and application of platelet activating factor to neuronal cultures activated GSK-3β [47]. These ﬁ ndings suggest that GSK-3β dysregulation could contribute to HIV-induced neuronal apoptosis. Parkinson’s disease is an age-related disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra pars compacta. A similar loss in nigral-striatal dopaminergic neurons is produced by intracerebral administration of the catecholaminergic neurotoxin 6hydroxydopamine [48], and it is widely used to model this neurodegenerative disorder. Chen et al. [49] have recently reported that 6-hydroxydopamine activates GSK-3β (ser9 dephosphorylation) in cultured CNS neurons. Focal ischemia results in a loss of cortical neurons and represents an acute model of neurodegeneration. Phosphorylation of GSK-3β on tyr216 leads to increased enzyme activity and cell death [40]. In rats, tyr216 phosphorylation was increased in degenerating cortical neurons induced by ischemia [40], suggesting that tyr216 phosphorylation represents an important mechanism by which cellular insults can lead to cell death. 9.3 GSK-3 INHIBITORS AND NEUROPROTECTION 9.3.1 Lithium As mentioned earlier, GSK-3 is inhibited by PKB-mediated phosphorylation [9], which may play a critical role in regulating neuronal cell survival [7,8]. Recombinant gene overexpression studies using active or dominant-negative

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proteins indicate that GSK-3 can play a central role in the survival of some neuronal cell populations in response to apoptotic triggers [11,12,16,40]. However, interpretations based on recombinant genetic studies alone need to be treated with caution, given the possible artefactual perturbation of normal patterns of the temporal and spatial regulation of protein kinase-mediated signaling complexes. It is becoming apparent that cellular responses to GSK-3 modulation can vary according to the nature of the inhibitory signal [50,51]. Thus the cellular consequences of GSK-3 inhibition may vary according to cell type and range of environmental stimuli. It should also be noted that given the potential degree of cross-talk and dynamics of GSK-3 regulation, the impact of overexpressing recombinant mutant GSK-3 and/or protein inhibitors of GSK-3 activity toward some substrates may be distinct from pharmacological inhibition of endogenous pools of active GSK-3 in some cell types. Lithium is an ATP noncompetitive low (mM) inhibitor of GSK-3 activity [52]. Lithium has been used extensively as a tool to support a role for GSK-3 in neuropathological settings. Several studies have shown that lithium inhibits Tau hyperphosphorylation, degeneration and cognitive deﬁcits in animal models [31,53–55], and can protect cultured neurons against Aβ-induced neurotoxicity [38]. Interestingly, therapeutic concentrations of lithium have been claimed to reduce production of Aβ peptides in a mouse model of AD, by interfering with amyloid precursor protein cleavage [56]. Lithium may ameliorate also neurotoxicity in vivo of the HIV-gp120 envelope protein [57] and prion peptide in neuronal cell culture [45]. Neuroprotection against glutamate excitotoxicity in dissociated cell cultures [58] and kainic acid-induced neurotoxicity in hippocampal slice cultures [59] have been described for lithium in animal models of excitotoxin injury (e.g., cerebral ischemia and quinolinic acid infusion) [58]. Any interpretation of lithium action is complicated by the many additional activities of this compound, including inhibition of polyphosphate 1-phosphatase, inositol monophosphatase, casein kinase II, mitogen-activated protein kinase-activated protein kinase 2, and p38-regulated/activated kinase [60,61], as well as the activation of PI 3-kinase/PKB and c-Jun N-terminal kinase in cellular assays [62,63].

9.3.2

Small-Molecule Inhibitors

More than 30 inhibitors of GSK-3 have been identiﬁed to date [64]. Seven of these have been co-crystallized with GSK-3β and all localize within the ATP binding pocket of the enzyme. Only a limited number of these molecules have actually been applied as neuroprotective agents until now. Novel potent and selective small-molecule ATP-competitive inhibitors of GSK-3 activity have recently been described [65]. SB-216763 and SB-415286 (Fig. 9.1) are structurally distinct maleimides that inhibit puriﬁed GSK-3α

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H N

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Chemical structure of known GSK-3β inhibitors.

activity in vitro with IC50 s of 78 nM and 74 nM, respectively, and show similar potency toward puriﬁed GSK-3β. Both compounds are highly selective inhibitors, exhibiting no signiﬁcant activity toward any member of a panel of 24 other related protein kinases, including PKB and 3-phosphoinositidedependent protein kinase-1 [65]. SB-216763 and SB-415286 are potent promoters of the survival of cerebellar granule neurons and sensory ganglionic neurons following trophic factor withdrawal, or inhibition of PI 3-kinase activity with LY294002 [66], the latter inactivating the PI 3-kinase/PKB survival pathway, thereby activating GSK-3β [67] (Fig. 9.2). The inhibition of neuronal cell death mediated by these compounds correlated with inhibition of GSK-3 activity and phosphorylation or stabilization of the GSK-3 substrates Tau and β-catenin, respectively, in intact neurons [66]. Rescue of cerebellar granule neurons from low potassium-induced apoptosis by SB415286 has been conﬁ rmed by others [68]. These data provide clear pharma-

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SB-415286 (mM) Figure 9.2 The GSK-3 inhibitor SB-415286 protects cerebellar granule neurons from death induced by: (A) survival factor withdrawal; (B) PI 3-kinase inhibition (LY294002).

cological and biochemical evidence that selective inhibition of endogenous GSK-3 pools in primary neurons is sufﬁcient to prevent death, implicating GSK-3 as a physiologically relevant principal regulatory target of the PI 3kinase/PKB neuronal survival pathway. Agents that increase intracellular cyclic AMP levels, such as the corticotropin-releasing factor and related peptides, were found to confer neuroprotection of LY294002-injured cerebellar granule neurons via cyclic AMP-dependent phosphorylation (and inactivation) of GSK-3 [67]. Excessive glutamatergic neurotransmission, particularly when mediated by the N-methyl-d-aspartic acid (NMDA) subtype of glutamate receptors, is thought to underlie neuronal cell death in several types of acute brain injury, including cerebral ischemia, hypoglycaemia, seizures, and mechanical trauma [69]. Glutamate-induced neuronal cell death in discrete brain areas has also

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been implicated in chronic neurodegenerative disorders such as AD, Parkinson’s disease, and Huntington’s chorea [70,71]. Long-term treatment with SB216763 and SB-415286 protected cultured CNS neurons against excitotoxic death mediated by both NMDA and non-NMDA receptors [72] (Fig. 9.3). Interestingly short preincubation or co-treatment alone with GSK-3 inhibitor was insufﬁcient to render CNS neurons resistant to excitotoxin injury [72], in contrast to cerebellar granule neuron death caused by pharmacological or physiological block of the PI 3-kinase/PKB cascade [66]. This suggests that distinct mechanisms may underlie these different death paradigms. The molecular basis for the neuroprotective effects of persistent treatment with GSK-3 inhibitors is not known but may reﬂect, in part, enhanced gene expression through reduced phosphorylation of c-Jun, causing disinhibition of the DNA binding activity of c-Jun [73]. Another GSK-3β inhibitor, Chir025, has been reported [74] to reduce (1) cultured hippocampal neuron death following glutamate exposure by 15–20% versus vehicle-treated controls, (2) cultured cortical neuron death following oxygen-glucose deprivation by about 50%, and (3) infarct size following focal cerebral ischemia by nearly 20%. Protection in the stroke model did not appear to be mediated by anti-apoptotic effects, but was associated with increases in the neuroprotective protein Bcl-2 [74]. Chir025 is claimed to act as a simple competitive inhibitor of ATP (IC50 = 27 nM for GSK-3β, no data for GSK-3α) and to be structurally similar to the substituted aminopyrimidine derivatives previously described by Ring et al. [75]. However, the structure of Chir025 was not disclosed [74,75].

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SB-415286 (mM) Figure 9.3 Prolonged pre-treatment of hippocampal neurons with the GSK-3 inhibitor SB-415286 reduces death caused by glutamic acid. Hippocampal cell cultures were prepared from rat embryonic day 18 and incubated with SB-415286 at day 1. Cultures at day 9 were challenged with glutamic acid (100 μM for 30 min) in the absence of SB-415286. Neuronal cell viability was quantiﬁed 24 h later by measuring release of lactate dehydrogenase (LDH). Values are means ± SD (n = 3). *p < 0.05 and # p < 0.005 vs glutamate.

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Huntington’s disease is a neurodegenerative disorder caused by polyglutamine extension of the huntingtin protein [76]. The modiﬁed protein is toxic and forms intracellular aggregates. Decreased β-catenin levels and reduced transcription in cells that express mutant huntingtin indicates that GSK-3 modulates cellular toxicity. Indeed, the GSK-3 inhibitor SB-216763 or overexpression of a dominant-negative GSK-3β protected cells from the toxicity of mutant huntingtin [77]. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder characterized by the preferential loss of upper motor neurons of the motor cortex and lower motor neurons of the brainstem and spinal cord. The precise pathogenic mechanisms of motoneuron death have not yet been clearly established. However, point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial ALS. Koh et al. [78], using VSC4.1 motoneuron cells transfected with G93A- or A4Vmutant types of hSOD1, showed both a greatly reduced cellular viability and activation of GSK-3β. Treatment of G93A and A4V cells with a GSK-3β inhibitor {2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxidiazole} [79] reduced these mutation-induced motor neuronal cell deaths by activating heat shock transcription factor-1 and by reducing cytochrome c release, caspase-3 activation and poly(ADP-ribose) polymerase cleavage [78]. AR-A014418 is a thiazole, N-(4-methoxybenzyl)-N′-(nitro-1,3-thiazol-2yl) urea that is a selective and potent inhibitor of GSK-3 (IC50 = 104 ± 27 nM), in an ATP-competitive manner (K i = 38 nM) [80] (Fig. 9.1). AR-A014418 is speciﬁc against GSK-3 in a panel of 26 kinases [80]. AR-A014418 inhibited Tau phosphorylation at a GSK3-speciﬁc site (ser396) in cells stably expressing human four-repeat Tau protein [80]. AR-A014418 protected N2A neuroblastoma cells against death mediated by pharmacological inhibition of the PI 3-kinase/PKB survival pathway with LY294002. Furthermore AR-A014418 inhibited neurodegeneration mediated by Aβ peptide in hippocampal slices [80]. In transgenic mice overexpressing mutant human Tau, treatment with AR-A014418 resulted in a signiﬁcant reduction in aggregated Tau and degeneration [55]. Overexpression of wild-type human Tau in Drosophila disrupts axonal transport, causing vesicle aggregation that is associated with loss of locomotor function [54]. AR-A014418 reversed both the axonal transport and locomotor phenotypes in this model [54], whereas co-expression of constitutively active GSK-3β enhanced the pathological effects of Tau overexpression. These data show that Tau abnormalities are capable of disrupting neuronal cell function, before the classical pathological hallmarks are evident, and suggest a role for GSK-3 in tauopathies.

9.4

CONCLUDING REMARKS

Based on knowledge of the literature, several therapeutic areas might beneﬁt from the development of GSK-3 inhibitors [64], including neurodegenerative

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disorders. Indeed, the two main diagnostic features of AD, β-amyloid toxicity/aggregation and Tau hyperphosphorylation/aggregation, are both directly linked to GSK-3, and this is one of the enzymes responsible for the appearance of AD-speciﬁc epitopes in Tau. The existence of three closely related, noninterchangeable isoforms of GSK-3 (GSK-3α, GSK-3β, and GSK-3β2), the multiple, sometimes apparently opposing, functions of GSK-3, and the limited characterization of selectivity, cell permeability, and stability of pharmacological inhibitors of GSK-3 mean that the development of therapeutically useful anti–GSK-3 drugs will not be easy. In addition the toxicity, associated side effects, and concerns regarding the absorption, distribution, metabolism, and excretion of these inhibitors affect their clinical potential. Clearly, much remains to fully address these issues, including a more complete understanding of the consequences of inhibiting components of the multiprotein complex of which GSK-3 is a part.

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and beta-adrenergic agonists in rat epididymal fat cells: Activation of protein kinase B by wortmannin-sensitive and -insensitive mechanisms. J. Biol. Chem., 272, 7713–7719. Pap M., Cooper G.M. (1998). Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem., 273, 19929–19932. Hetman M., Cavanaugh J.E., Kimelman D., Xia Z. (2000). Role of glycogen synthase kinase-3β in neuronal apoptosis induced by trophic withdrawal. J. Neurosci., 20, 2567–2574. Bijur G.N., De Sarno P., Jope R.S. (2000). Glycogen synthease kinase-3β facilitates staurosporine- and heat shock-induced apoptosis: Protection by lithium. J. Biol. Chem., 275, 7583–7590. Woodgett J.R. (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J., 9, 2431–2438. Delcommenne M., Tan C., Gray V., Rue L., Woodgett J., Dedhar S. (1998). Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc. Nat. Acad. Sci. USA, 95, 11211–11216. Kobayashi T., Cohen P. (1999). Activation of serum- and glucocorticoidregulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J., 339, 319–328. Crowder R.J., Freeman R.S. (2000). Glycogen synthase kinase-3β activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J. Biol. Chem., 275, 34266–34271. Frame S., Cohen P. (2001). GSK3 takes centre stage more than 20 years after its discovery. Biochem. J., 359, 1–16. Selkoe D.J. (1997). Alzheimer’s disease: genotypes, phenotype and treatments. Science, 275, 630–631. Selkoe D.J. (2001). Alzheimer’s disease: Genes, proteins, and therapy. Physiol. Rev., 81, 741–766. Lee V.M., Goedert M., Trojanowski J.Q. (2001). Neurodegenerative tauopathies. An. Rev. Neurosci., 24, 1121–1159. Ferrer I., Barrachina M., Puig B. (2002). Glycogen synthase kinase-3 is associated with neuronal and glial hyperphosphorylated Tau deposits in Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy and corticobasal degeneration. Acta Neuropathol., 104, 583–591. Shiurba R.A., Ishiguro K., Takahashi M., Sato K., Spooner E.T., Mercken M., Yoshida R., Wheelock T.R., Yanagawa H., Imahori K., Nixon R.A. (1996). Immunocytochemistry of tau phosphoserine 413 and Tau protein kinase I in Alzheimer pathology. Brain Res., 737, 119–132. Pei J.J., Braak E., Braak H., Grundke-Iqbal I., Iqbal K., Winblad B., Cowburn R.F. (1999). Distribution of active glycogen synthase kinase 3β (GSK-3β) in brains staged for Alzheimer disease neuroﬁbrillary changes. J. Neuropathol. Exp. Neurol., 58, 1010–1019.

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25. Mandelkow E.M., Drewes G., Biernat J., Gustke N., Van Lint J., Vandenheede J.R., Mandelkow E. (1992). Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. FEBS Lett., 314, 315–321. 26. Leroy K., Boutajangout A., Authelet M., Woodgett J.R., Anderton B.H., Brion J.-P. (2002). The active form of glycogen synthase kinase-3β is associated with granulovacuolar degeneration in neurons in Alzheimer’s disease. Acta Neuropathol., 103, 91–99. 27. Lovestone S., Reynolds C.H., Latimer D., Davis D.R., Anderton B.H., Gallo J.-M., Hanger D., Mulot S., Marquardt B., Stabel S., Woodgett J.R., Miller C.C.J. (1994). Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr. Biol., 4, 1077–1086. 28. Lucas J.J., Hernández F., Gómez-Ramos P., Morán M.A., Hen R., Avila J. (2001). Decreased nuclear β-catenin, Tau hyperphosphorylation and neurodegeneration in GSK-3β conditional transgenic mice. EMBO J., 20, 27–39. 29. Hernández F., Borrell J., Guaza C., Avila J., Lucas J.J. (2002). Spatial learning deﬁcit in transgenic mice that conditionally over-express GSK-3β in the brain but do not form tau ﬁ laments. J. Neurochem., 83, 1529–1533. 30. Jackson G.R., Wiedau-Pazos M., Sang T.-K., Wagle N., Brown C.A., Massachi S., Geschwind D.H. (2002). Human wild-type Tau interacts with wingless pathway components and produces neuroﬁbrillary pathology in Drosophila. Neuron, 34, 509–519. 31. Liu S.J., Zhang A.H., Li H.L., Wang Q., Deng H.M., Netzer W.J., Xu H., Wang J.Z. (2003). Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of Tau and impairment of spatial memory. J. Neurochem., 87, 1333–1344. 32. Hernández F., Pérez M., Lucas J.J., Mata A.M., Bhat R., Avila J. (2004). Glycogen synthase kinase-3 plays a crucial role in tau exon 10 splicing and intranuclear distribution of SC35: Implications for Alzheimer’s disease. J. Biol. Chem., 279, 3801–3806. 33. Takashima A., Noguchi K., Michel G., Mercken M., Hoshi M., Ishiguro K., Imahori K. (1996). Exposure of rat hippocampal neurons to amyloid β peptide (25–35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of Tau protein kinase I/glycogen synthase kinase-3β. Neurosci. Lett., 203, 33–36. 34. Kim H.-S., Kim E.-M., Lee J.-P., Park C.H., Kim S., Seo J.-H., Chang K.-A., Yu E., Jeong S.-J., Chong Y.H., Suh Y.-H. (2003). C-terminal fragments of amyloid precursor protein exert neurotoxicity by inducing glycogen synthase kinase-3β expression. FASEB J., Express Article doi: 10.1096/fj.03-010fje. 35. Hoshi M., Sato M., Matsumoto S., Noguchi A., Yasutake K., Yoshida N., Sato K. (2003). Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate Tau protein kinase I/glycogen synthase kinase-3β. Proc. Nat. Acad. Sci. USA, 100, 6370–6375. 36. Takashima A., Noguchi K., Sato K., Hoshino T., Imahori K. (1993). Tau protein kinase I is essential for amyloid β-protein-induced neurotoxicity. Proc. Nat. Acad. Sci. USA, 90, 7789–7793.

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10 PROTEIN KINASE ASSAYS FOR DRUG DISCOVERY Miguel Medina, Ana Fuertes, Ester Martín-Aparicio, María del Monte-Millán, María-Luisa Navarro, and María José Pérez-Puerto Neuropharma S.A., Tres Cantos, Madrid

Contents 10.1 Protein Kinases as Drug Targets 10.2 GSK-3 Inhibitors as Therapeutic Agents 10.3 In Vitro Protein Kinase Activity Assays 10.3.1 Radioactivity-Based Assays 10.3.2 Fluorescence-Based Assays 10.3.3 Luminiscence-Based Assays 10.3.4 Binding Assays and Virtual Screening 10.4 Functional Cell-Based Assays for GSK-3 Inhibition 10.5 The Selectivity Issue 16.6 Concluding Remarks

10.1

PROTEIN KINASES AS DRUG TARGETS

Protein phosphorylation is recognized as one of the most signiﬁcant signal transduction mechanisms by which extracellular signals regulate most critical cellular processes. For discovery in the 1950s of reversible protein phosphorylation as a biological regulatory process, Edmond H. Fischer and Edwin G. Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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Krebs were awarded the 1992 Nobel Prize for Physiology and Medicine. The importance of protein phosphorylation as a regulatory mechanism is revealed by the fact that over 30% of intracellular proteins can be phosphorylated at one or more sites. Since the discovery of the ﬁrst protein kinases (i.e., the casein kinases and phosphorylase kinase) some 50 years ago [1], it has become clear that phosphorylation not only regulates enzymatic activity but also the function of structural proteins through conformational and charge effects, and more recently it has been found that protein-linked phosphates can act as binding sites for other proteins, with a sound impact on cell signaling [2]. Complete sequencing of the human genome has now revealed a total of 518 predicted protein kinase genes (1.7% of all genes) [3], about half the number predicted some 20 years ago [4], representing the largest enzyme family of the human genome. It has to be considered however that the actual total number of protein kinase gene products is surely much greater than 518, due to the expression of alternatively spliced forms, which are already well known for many protein kinases. On the other side of the coin, we do have to keep in mind that the phosphorylation status of a protein and its phosphorylation-dependent regulatory function depends on a balance between the protein kinases and the protein phosphatases that remove the phosphate moities. In combination, the protein kinase and phosphatase genes (the so-called kinome and phosphatome, respectively) account for nearly 2.5% of all genes in most eukaryotic species [5]. Disruption of protein kinase function or regulation is involved in a host of major diseases, including cancer and autoimmune, inﬂammatory, cardiovascular, metabolic, and neurological diseases. Furthermore, defects in genes encoding for protein kinases underlie a number of inherited and acquired disorders, including leukemias, lymphomas, and autoimmune diseases. As a result, protein kinases have been considered as excellent candidates for therapeutic intervention. This interest in protein kinases as targets for therapeutic intervention has been recently fueled by the approval of a variety of kinase-directed smallmolecule drugs for cancer treatment, including GleevecTM (Imatinib, an inhibitor of the tyrosine kinase BCR-Abl), IressaTM (Geﬁtinib, an EGFR tyrosine kinase inhibitor), HerceptinTM (Trastuzumab, a humanized mouse antibody against the tyrosine kinase receptor HER2), and AvastinTM (Bevacizumab, a recombinant humanized antibody against VEGF) [reviewed in 6], which have come to show that small molecule protein kinase inhibitors can indeed be tailored to address speciﬁc diseases. As a consequence, there has been an increase in the amount of research and development spent on kinases, primarily in the areas of cancer, immune diseases, diabetes, and, most recently, in neurological disorders. Biotech and pharmaceutical companies are gearing up to capitalize on creating new targeted therapeutics. There are currently over 50 kinase-targeted drugs in clinical development and many more in various stages of preclinical development. It is estimated that protein kinases

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currently account for about 25% to 30% of all efforts in drug development programs, becoming the major drug discovery targets of the twenty-ﬁ rst century [7].

10.2

GSK-3 INHIBITORS AS THERAPEUTIC AGENTS

Glycogen synthase kinase 3 (GSK-3) was identiﬁed a quarter of century ago as a protein kinase that inhibits glycogen syntyhase (GS), the enzyme that catalyzes the transfer of glucose from UDPG to glycogen [8]. A large body of evidence has since then accumulated to show that GSK-3 plays a central role in signal transduction pathways involved in cell survival triggered by insulin and other growth factors as well as in embryonic development through the Wnt pathway (reviewed in [9]). Since the mid-1990s there has been a near exponential rise in the level of GSK-3-related research focusing on its implication in apoptosis, type 2 diabetes, cancer, bipolar disorders, stroke, and neurodegenerative diseases. As a consequence the therapeutic potential of GSK3 inhibitors has become a major area of pharmaceutical interest (reviewed in [10]). Nearly half a century after the discovery that lithium salts were effective in treating manic-depression illness, GSK-3 was discovered as one of lithium’s main targets [11]. Since then lithium has been widely used as a pharmacological inhibitor of GSK-3 [12]. By 2004 more than 30 small organic molecules had been already reported as GSK-3 inhibitors, some with IC50 values in the nanomolar range [13]. Some of these inhibitors have also been co-crystallized with GSK-3β, which have provided a detailed understanding of the 3D structure of the ATP binding pocket and its interaction with the inhibitor molecules and set the basis for structure-based drug design programs. The emergence of the ﬁeld is reﬂected by the considerable patent and basic activity reported in the last few years in search for novel, potent, and more selective GSK3 inhibitors.

10.3

IN VITRO PROTEIN KINASE ACTIVITY ASSAYS

Mostly three technologies are used for hit-and-lead ﬁnding in modern protein kinase drug discovery: high-throughput screening (HTS) of random libraries, three-dimensional structure-based drug design based on X-ray data, and focused libraries around limited number of new cores. In any case, in order to discover and develop novel kinase inhibitors, it is imperative to develop enzyme assays speciﬁc for individual kinases that allow the screening of chemical libraries and identifying inhibitors that meet the requirements of a therapeutic drug. Many different approaches can be taken to develop assays for screening of potential kinase inhibitors, and issues such as the size of the compound library to be screened, the required throughput, and cost must be considered. The basic components of any kinase activity assay are simple: a

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puriﬁed kinase, substrate, and ATP. However, which substrate is used and how the phosphate is incorporated on it during detection of the enzymatic reaction contribute to the variation of assays found in the literature. If the assay is to be used in a high-throughput primary screening program, issues such as the Z value, ﬂexibility, equipment, and cost are key aspects, and the assay should be not only speciﬁc but also scalable to a high-throughput level in order to utilize it in screening thousands to millions of compounds. On the other hand, if the assay is to be used in early-stage protein characterization, secondary screening, or lead optimization, then the considerations might be fewer and/or different. 10.3.1

Radioactivity-Based Assays

The high sensitivity achieved with radioactivity detection were at the basis of the ﬁ rst kinase activity assays some 40 years ago, and radiometric assays are still widely used because of that and the fact that they are not limited by the substrate being assayed. The principle of the method is very simple, and the assay is straightforward. The assay measures direct transfer of the radiolabeled phosphate moiety to the substrate, and it is not affected by other binding events. The classical method consist in incubating together the puriﬁed kinase enzyme, a peptide substrate and [γ-32P]ATP in the appropriate buffer and temperature. After incubation the mix is ﬁltered through a membrane that traps the substrate but not free [γ-32P]ATP. Quantitation of the radioactivity remained on the ﬁ lter by gamma-scintillation counting allows for determination of substrate phosphorylation levels (Fig. 10.1). Innovations of this classical method had occurred over the years. For instance, the widespread use of softer, higher half-time beta-emitting radioisotope [33P] has come to replace [32P]. In addition, other high-afﬁ nity binding matrices (e.g., biotin capture membranes) have been developed. A one-step, no wash assay variant of the classical radioactive method based on a scintillation proximity assay (SPA) has also been developed, and it is widely used. In this assay the substrate is biotinylated allowing it to bind to streptavidincoated scintillation beads (Fig. 10.2). When the substrate is phosphorylated, the proximity of the radioactivity to the beads causes a greater emission signal. Unbound, free [γ-33P]ATP does not excite the SPA bead and does not need to be washed away. In the particular case of GSK-3, there are several commercial sources of puriﬁed recombinant enzyme (whether the α or β isoform), even some commercial assay kits. As for the substrate, the most widely used are the ones derived from the sequence of the human glycogen synthase. More speciﬁcally, since GSK-3 has a much higher activity on pre-phosphorylated (“primed”) substrates, the phospho-GS2 peptide [YRRAAVPPSPSLSRHSSPHQS(p)E DEEE] is the best choice. Despite a continuous trend in recent years to move away from radiactivity-based kinase activity assays because of safety and cost concerns

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A

wash 33

P

Substrate peptide

Filter membrane

[γ-33P]-ATP Kinase + Mg++

Scintillation counting

Unbound [γ-33P]-ATP

B

Reaction mix: Kinase + [γ-33P]-ATP + Mg++

33

P -ATP

33

P

B S

No activity, no signal

B S

Kinase activity

Figure 10.1 Radiometric assays. The most common method consists in incubating together a puriﬁed protein kinase, a peptide substrate, and [γ-33P]-ATP in the appropriate buffer and temperature. After incubation, the reaction mix is ﬁ ltered through a substrate-trapping membrane and the radioactivity remained in the membrane quantitated by scintillation counting (A). A variant of the classical method uses a solid-phase assay in which a biotinylated peptide is bound to strepatividincoated plates (B) and the kinase reaction is performed directed onto the well. See color plates.

as well as strict regulations regarding radioisotopes handling and disposal, this type of assays is still widely used. 10.3.2

Fluorescence-Based Assays

The desire to avoid the use of radioisotopes has spurred the development of kinase activity assays in which the ﬁ nal readout is based on ﬂuorescence signals [14]. The ﬁ rst of these methods is an antibody-based kinase assay in which the puriﬁed kinase, a peptide substrate, and ATP are incubated together with an antibody that speciﬁcally recognizes the peptide substrate only in its phosphorylated form. After incubation, as in an ELISA assay, the unbound antibody will be washed out and the antibody-antigen complexes can be detected by ﬂuorescence or a colorimetric assay with horseradish peroxidase. The biggest, obvious limitation of this assay is its absolute dependence on the availability of phospho-speciﬁc antibodies, either general

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A

Bound radiolabel Free radiolabel

P

33 33

P

Energy absorbed by medium Bead stimulated to emit light

B

33

Biotinylated peptide

[g-33P]-ATP

P

B

B Kinase + Mg++ SPA beads

Figure 10.2 Scintillation proximity assay (SPA). The basic concept of the SPA assay is that when a radioactive molecule is held in close proximity to a SPA scintillation bead (or nanoparticle), the scintillation contained into the bead is stimulated to emit light (A), which can be detected on a scintillation counter. In contrast, if the radioactive molecule is free in the solution, it will not have enough energy to reach the beads and no light will be emitted. Tipically kinase SPAs are conﬁgured using streptavidincoated beads and biotinylated substrated (B). See color plates.

antiphosphotyrosine or -phosphoserine/threonine antibodies, or custom ones against the peptide substrate. Most kinase assays are geared for high-throughput screening (HTS) applications in which chemical libraries consisting of thousands of compound must be screened in search for kinase inhibitors. In recent years there has been a shift to use nonradiometric, ﬂuorescence-based kinase activity assays, specially for HTS applications. At least three different types of ﬂuorescent assays are currently available and commonly used, based on ﬂuorescence polarization (FP), Forster resonance energy transfer (FRET), and time-resolved FRET (TR-FRET). Polarization is a general property of ﬂuorescent molecules. Dye molecules with their absorption transition vectors aligned parallel to the electric vector

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of linearly polarized light are selectively excited. When a ﬂuorescent dye is attached to a small, rapidly rotating molecule, the initially photoselected orientational distribution becomes randomized prior to emission, resulting in low FP. Conversely, binding of the low molecular weight tracer to a large, slowly rotating molecule results in high ﬂuorescence polarization. Therefore, FP provides a direct readout of the extent of tracer binding to proteins, nucleic acids, and other biopolymers [15]. FP kinase assays are based on measuring changes in polarization of light emitted by ﬂuorescently labeled peptides by taking advantage of the selective binding of the phospho-peptide to speciﬁc antiphospho-antibodies or to a trivalent metal cation bound to beads or nanoparticles. The rotation of the phosphorylated peptide substrate will be slowed by the bound beads resulting in more polarization of the emitted light. Forster resonance energy transfer (FRET) is a well-known spectroscopic technique for monitoring changes in the proximity and mutual orientation of pairs of chromophores. It has long been used in biochemistry and cell biology to assess distances and orientations between speciﬁc labeling sites within a single macromolecule or between two separates molecules [16]. FRET involves the nonradioactive transfer of excitation energy from an excited donor ﬂuorophore to a proximal ground-state acceptor ﬂuorophore. When applied to measuring kinase activity, FRET-based assays are based on the differential sensitivity of phosphorylated versus nonphosphorylated peptides to proteolytic cleavage. Substrate peptide is labeled with two different ﬂuorophores and proteolytic cleavage disrupts FRET between the donor and acceptor ﬂuorophores. Reaction progress is then quantitated by calculating the emission ratio of donor to acceptor after excitation of the donor ﬂuorophore at the appropriate wavelength. The main drawback of FP- and FRET-based kinase assays, especially for screening applications, is autoﬂuorescence from the tested compounds, which makes it hard to separate signal from noise during the assay. To avoid this problem, FRET-peptides that ﬂuoresce at wavelengths other than those present in most chemical libraries have been developed. In an attempt to minimize this compound’s interference problem, TR-FRET measurements are emerging as one of the preferred screening assay formats in drug discovery laboratories and have also been adapted to kinase activity assays [17,18]. TR-FRET-based assays take advantage of the fact that some rare earth compounds, such as europium and terbium, ﬂuoresce later than regular ﬂuorophores. Peptide substrate labeled with terbium or europium as the donor and a regular ﬂuorophore as the acceptor can then be used in a kinase reaction. When the donor and the acceptor molecules are brought into proximity, energy transfer takes place, causing an increase in acceptor ﬂuorescence and a decrease in donor ﬂuorescence. These signals can then be read in a timeresolved manner to reduce assay interference due to color quenchers, light scatterants, or autoﬂuorescent compounds.

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10.3.3

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Luminiscence-Based Assays

Another nonradiometric format of kinase activity assays that has become popular in recent years is that based on luminiscence. In this case kinase activity measured by quantitating changes in ATP levels during the reaction uses luciferase. The luminiscent reaction catalyzed by luciferase converts luciferin into oxyluciferin and light. During the kinase reaction ATP is depleted, resulting in a decrease of emitted light that can be quantitated by a luminometer. The main advantages of luminiscence-based assays are that no radioactivity is involved, no antibody is required, and it is not restricted to peptide substrates. However, a serious drawback is that it loses linearity when high ATP concentrations are present in the reaction mix because of the high afﬁ nity of luciferase for ATP.

10.3.4

Binding Assays and Virtual Screening

Unlike classical functional kinase assays, which are based on detecting the phosphorylation of the substrate by the enzyme, binding assays measure direct binding of compounds to the kinase and determine potency by estimating binding afﬁ nities. Because most kinase inhibitors act by binding to the kinase ATP binding pocket and competing with ATP [19], a binding assay can be designed in which the compound binding to the kinase ATP binding site can be measured by competition with a conjugated binding probe. Binding can then be quantitated by biophysical methods or biochemically by coupling it to an enzymatic signal ampliﬁcation system and a colorimetric or ﬂuorescent readout. Binding to sites different than the ATP binding site (substrate site or allosteric sites) can, of course, result also in enzyme inhibition and in some cases might be preferred to avoid ATP competition. Normally allosteric inhibitors are searched for in traditional kinase activity assays by using high ATP concentrations because they are more likely to be selective for the kinase they inhibit (see below for comments on selectivity), although they are more difﬁcult to ﬁ nd. Thiadiazolidinones (TDZD) are the only GSK-3 inhibitors described so far to be non–ATP competitors [20]. The surge in recent years of molecular modeling techniques and the accumulation of crystallographic-based three-dimensional structures of many protein kinases, including GSK-3 [21]), have allowed the use of virtual screening (three-dimensional structure-based drug design based on X-ray data) in locating the novel small molecules that bind and inhibit protein kinases [22]. Virtual screening is used to determine which small molecules theoretically have the potential to bind to a given protein. Databases of thousands of small molecules are used to evaluate the potential of the small molecules they contain [23]. To discern which small molecules will bind to a protein kinase, the library of compounds is screened against a panel of proteins, and the binding afﬁ nity for each compound is determined. The potential of a ligand for binding can be assessed by considering its kinase

FUNCTIONAL CELL-BASED ASSAYS FOR GSK-3 INHIBITION

197

structure, charge distribution, energy minimization through potential aromatic pi-stacking, reduction of steric repulsion, and dynamic simulations [24]. By determining the structure of the small molecule and the protein, and by considering their potential steric and electrostatic interaction, it is possible to predict whether a ligand will bind in an assay [24]. “Guided docking” is the term used for this approach because it uses chemical information to guide the orientation of the small molecule into the binding site [25]. Generally, the ligand has potential to become a drug if it binds at certain sites and inhibits activity. However, binding ligands to the substrate binding site is becoming a more common approach. Possible inhibitors detected by virtual screening need obviously to be experimentally tested in a kinase activity assay.

10.4 FUNCTIONAL CELL-BASED ASSAYS FOR GSK-3 INHIBITION After primary screening in any of the in vitro kinase activity assays described above, a cell-based assay is used to test the small molecules’ activity with actual cells, testing at the same time for cell permeability and potential effects on cell viability. Cell-based assays can conﬁ rm the activity of agents tested in target-based screening assays and can assess the agent’s pharmacologic effects [26]. Furthermore, recent advances in proteomics and protein array technologies, combined with the use of phospho-speciﬁc antibodies for a great variety of cellular proteins, have made it possible to detect speciﬁc phosphorylation events in cells and tissues [27,28]. Cell-based assays for detecting speciﬁcally GSK-3 activity have been described over the years through readouts that detect GSK-3-dependent phosphorylation events or their cellular consequences. Brieﬂy, we will mention the most common ones, but with the caveat that the choices depend on many factors, including the therapeutic area of interest and personal preference. First of all, GSK-3 inhibitors can be tested in cells by determining their effect on glycogen synthase (GS) activity in CHO-IR expressing the human insulin receptor, primary hepatocytes, or isolated skeletal muscle preparations [29]. The GS activity ratio is measured by the GS activity in the absence of glucose-6-phosphate divided by the GS activity in the presence of 5 mM glucose-6-phosphate according to the classic method [30]. Availability of antibodies that speciﬁcally recognize a GSK-3 substrate only when it is phosphorylated has also made it possible to detect GSK-3 activity in cultured cells. One of those substrates is the neuronal-speciﬁc microtubule-associated protein Tau, which plays a central role in Alzheimer’s disease pathology. Phosphorylation at residue Ser396 is thought to be speciﬁcally phosphorylated by GSK-3 and antibodies speciﬁc against this phosphoepitope are widely used to test for GSK-3 activity in cells and tissues either by western blot, ELISA, or immunohistochemistry [31,32].

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A less direct functional cell-based assay used for testing GSK-3 inhibitors is based in downstream effects of that inhibition. GSK-3β activity has been shown to be inhibited by the PI3K/PKB survival pathway, which normally supresses apoptosis [33]. Treatment of cells with a PI3K inhibitor such as LY294002 [34] inactivates that survival pathway, thereby activating GSK-3β and causing cell death. Protection from LY294002-induced cell death is thus a very useful way to test for GSK-3β inhibitors in cell-based assays [32]. Other cell-based assays used to test for the ability of selected compunds to actually inhibit GSK-3 in cells include TCF-dependent transcriptional activation of reporter genes such as luciferase [35]) or looking at β-catenin cytosolic levels, since both events are downstream of GSK-3β in the Wnt signaling pathway [36].

10.5

THE SELECTIVITY ISSUE

There has been a long debate as to whether protein kinases can be considered promising drug targets at all and whether selectivity between closely related protein kinases can be readily achieved. As for any protein kinase inhibitor, selectivity is an important indicator of pharmacological efﬁciency. Because the catalytic ATP binding site is conserved in many protein kinases and most protein kinase inhibitors target this site, speciﬁcity is a central issue [37,38]. Binding speciﬁcity and afﬁ nity cannot be readily predicted based on available sequence and structural information, and current proﬁ ling methods are limited by the difﬁculty of developing large number of kinase activity assays. However, in recent years some commercially available kinase proﬁ ling services allow for speciﬁcity studies in a large number of protein kinases. A limited kinase selectivity panel should be used to direct inhibitor series selection for medicinal chemistry programs immediately after conﬁrming hits in the primary screening assay and should be used throughout the lead optimization phase. The general implementation and use of such a kinase selectivity panel allows for the early availability of potency and selectivity data. Additionally, with the purity and structural information, these data will enable the chemistry of the large numbers of hits from primary screens to be prioritized and explored. Although protein kinase inhibitors selectivity is a key issue when used as a pharmacological tool to study signaling pathways or other cellular processes, absolute selectivity is not necessarily the best approach for therapeutic agents. High selectivity in treating complex diseases in which multiple pathways are deregulated can rapidly lead to resistance [13]. The selectivity of most of the available GSK-3 inhibitors is relatively poorly known and essentially based on their evaluation with rather limited kinases panels [13,38]. The structural relationship of GSK-3 with the catalytic ATP binding site of cyclin-dependent kinases (CDKs), in particular, CDK5 and CDK2, makes many of the GSK-3 inhibitors act also on those kinases. Because

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both GSK-3 isoforms α and β share a 98% sequence homology at the catalytic site, inhibitors targeting this site most likely will not not be able to differentiate between them. Such selectivity might require compounds acting at different sites of the enzyme. The unusual substrate speciﬁcity of GSK-3 provides an opportunity to develop highly selective, non-ATP competitive kinase inhibitors that target the priming phosphate required for phosphorylation of most substrates [9]. Some speciﬁc phosphorylated peptides targeting that site have been recently reported [39], but whether drug-like molecules can be developed that target this hydrophilic substrate binding site remains to be seen. 10.6

CONCLUDING REMARKS

Protein kinases are at the forefront of modern drug research. A relatively large number of compounds able to inhibit GSK-3 have been generated over the last few years. Some of them show high potency and a certain degree of speciﬁcity, but most of them bind to the ATP binding pocket and are competitive with ATP. Some of the compunds have recently shown activity in animal models of diabetes and nervous diseases. Novel approaches can now be used to generate drug-like GSK3 inhibitors that target different sites, such as the substrate binding site or allosteric sites. REFERENCES 1. Krebs E.G., Fischer E.H. (1955). Phosphorylase activity of skeletal muscle extracts. J. Biol. Chem., 216(1), 113–120. 2. Hunter T. (2000) Signalling—2000 and beyond. Cell, 100, 113–127. 3. Manning G., Whyte D., Martínez R., Hunter T., Sudarsanam S. (2002). The protein kinase complement of the human genome. Science, 298, 1912–1934. 4. Hunter T. (1987). A thousand and one protein kinases. Cell, 50, 823–829. 5. Hunter T., Manning G. (2003). Eukaryotic kinomes: genomic cataloguing of protein kinases and their evolution. In Bradshaw R.A., Dennis E.A., eds., Handbook of Cell Signaling, San Diego: Academic Press, 373–377. 6. Krause D.S., Van Etten R.A. (2005). Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med., 353(2), 172–187. 7. Cohen P. (2002). Protein kinases—The major drug targets of the twenty-ﬁ rst century? Nature Rev. Drug. Disc., 1, 309–315. 8. Embi N., Rylatt D.B., Cohen P. (1980). Glycogen synthase kinase-3 from rabbit skeletal muscle: Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem., 107(2), 519–527. 9. Cohen P., Frame S. ( 2001). The renaissance of GSK3. Nat. Rev. Mol. Cell. Biol., 2(10), 769–776. 10. Cohen P., Goedert M. (2004). GSK3 inhibitors: development and therapeutic potential. Nat. Rev. Drug Disc., 3(6), 479–487.

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11 ANIMAL MODELS WITH MODIFIED EXPRESSION OF GSK-3 FOR THE STUDY OF ITS PHYSIOLOGY AND OF ITS IMPLICATIONS IN HUMAN PATHOLOGIES Tobias Engel, Félix Hernández, Raquel Gómez-Sintes, Jesús Ávila, and José J. Lucas Centro de Biología Molecular “Severo Ochoa” CSIC/UAM, Madrid

Contents 11.1 Introduction 11.2 Mouse Models that Overexpress GSK-3 11.3 Mouse Models with Reduced GSK-3 Activity 11.4 Mouse Models with Modiﬁed Forms of GSK-3 11.5 Nonmurine Animal Models 11.6 Concluding Remarks

11.1

INTRODUCTION

Glycogen synthase kinase 3 (GSK-3) was initially identiﬁed more than three decades ago as an enzyme involved in the control of glycogen metabolism [1,2]. In mammals two closely related isoforms, GSK-3α and GSK-3β are present [3]. The more conserved isoform in evolution is GSK-3β with a wide expression throughout the animal kingdom, while GSK-3α is only found in Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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vertebrates [4]. GSK-3β is ubiquitously expressed in all tissues, with particularly abundant levels in the brain [5]. GSK-3 is now recognized as a key component of a large number of cellular processes and, when dysregulated, is thought to play a role in the etiology of various diseases. GSK-3 is involved in regulating cellular structure, function, and survival, and it is well established that it is implicated in several signaling pathways such as the insulin and insulin-like growth factor-1 (IGF-1) mediated signal transduction or the Wnt/Wingless signaling pathway. As mentioned, dysregulation of GSK-3 is proposed to be linked to several prevalent pathological conditions. These include diabetes and/or insulin resistance [6], muscle hypertrophy [7], cancer [8], bipolar mood disorder [9], schizophrenia [10], and neurodegenerative diseases such as Alzheimer’s disease (AD) [11] and Huntington’s disease [12,13]. In order to investigate its involvement in cellular processes and to clarify its implications in disease, a great effort is still being made to generate animal models that mimic pathological conditions through the dysregulation of GSK3. Up to date the main effort has been invested in creating animal models with altered expression of GSK-3, especially of the evolutionary well-conserved isoform GSK-3β. To achieve this goal, different strategies have been applied: overexpression, suppression and modulation of the expression of the enzyme GSK-3. Many of these models direct expression or modiﬁcation of GSK-3 to the central nervous system (CNS) for the generation of mouse models of AD. Nowadays several other species apart from the mouse are also used as model systems, which include Drosophila melanogaster and zebraﬁsh. Since the model system most widely used is the mouse, this review will focus primarily on the results obtained from studies in this species, but at the end of the chapter it will give a brief summary of the work performed in other organisms.

11.2

MOUSE MODELS THAT OVEREXPRESS GSK-3

One of the earliest diseases linked to dysfunctions of GSK-3β was AD. More recently it has been proposed that the deregulation of GSK-3β might affect other tauopathies like frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) [14]. Regarding AD, it has been demonstrated that GSK-3β mediates Tau hyperphosphorylation, β-amyloid-induced neurotoxicity and mutant presenilin-1 pathogenic effects [15]. Thus the ﬁ rst GSK-3 mouse models focused on the study of the implications of GSK-3β overexpression in the CNS on the development of AD and related neuropathological diseases. To achieve this goal, promoters with known neuronal expression like NF-L, thy-1, and the more region speciﬁc promoter CaMKIIα, were used. In 1997 Brownlees et al. generated the ﬁ rst two GSK-3 mouse models. One carried the human wild-type GSK-3β isoform as the transgene and the other one the mutant form of GSK-3β in which serine 9 of GSK-3β was mutated to

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205

alanine (S9A), thus supposedly resulting in a more active form by preventing inactivation through phosphorylation on Ser9 [5]). These transgenes were under the transcriptional control of either the ubiquitous murine sarcoma virus (MSV) promoter or the neuronal speciﬁc promoter of the murine neuroﬁ lament light chain (NF-L) [16]. Despite detecting the mRNAs of the two transgenes through reverse-transcribed PCR, no substantial increase in total activity of GSK-3β was observed, neither of the wild-type GSK-3β nor of the mutated form. They also observed that the expression of the mutated form of GSK-3β (S9A) was much lower, probably due to the fact of its increased activity. Despite no detectable GSK-3 overexpression, western blot analysis of the two mouse lines with the highest levels of transgenic GSK-3β activity revealed that the phosphorylation status of Tau was elevated at the AT8 epitope. The authors postulated that toxicity elicited by GSK-3β overexpression during embryonic and postnatal development of the CNS may explain why they were unable to generate mouse lines with detectable GSK-3β overexpression. The second transgenic animal published also expressed the S9A mutant form of the kinase, namely GSK-3β(S9A); this time under the control of the modiﬁed murine thy1 gene [17]. The engineered thy1 construct used in this model drives the expression of the transgene only postnatally and speciﬁcally in neurons. This mouse shows a two-fold increase in GSK-3β activity. Accordingly, an increase in phosphorylated Tau was demonstrated, but only in older transgenic mice (7–8 months). In a second round of characterization of this mouse line, Spittaels et al. also observed in these mice a signiﬁcant decrease in overall brain weight and volume, with the largest reduction occurring in the cerebral cortex. This reduction was not due to apoptosis, since there was no increase in TUNEL or in cleaved caspase-3 positive neurons [18]. Overexpression of GSK-3β in this model also has been shown to up- or downregulate the expression of numerous proteins in neurons, including downregulation of MAP2 [19]. Despite increased Tau phosphorylation and decreased brain weight these GSK-3β transgenic mice performed normally in the Morris water maze test [18]. One of the characteristics of persons affected by FTDP-17 is an increased expression of the longest human protein Tau isoform containing the four repeat regions in the carboxyl-terminal part [20]. For this reason Spittaels and co-workers in another approach cross-bred their GSK-3β (S9A) mice with transgenic mice that carry the longest human protein Tau isoform expressed under the same promoter (thy1). The authors observed a reduction of the number of axonal enlargements and motor impairments typical of these Tau transgenic animals [21]. In these mice an increase in phosphorylation of human Tau was demonstrated, although neither an increase in insoluble Tau aggregates nor the presence of paired helical ﬁ laments was observed. In a similar approach Li et al. used the mutant GSK-3β (S9A), resistant to inhibition by phosphorylation, under control of the neuron speciﬁc human platelet-derived growth factor (PDGF) β-chain promoter [22]. This promoter drove the expression mainly to the cortex and hippocampus of the transgenic

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brain. They observed an increase in GSK-3 activity without changes in the total level of GSK-3β. They also observed an increase in tau phosphorylation detected by the AT8 antibody by western blot analysis. In view of the postulated lethality of embryonic GSK-3β overexpression [16] as well as the known role of GSK-3β in development, a further GSK-3β transgenic mouse was generated [23] by using the conditional tetracyclineregulated system (as shown in Fig. 11.1A). In these mice transgene expression was under the control of the CaM kinase II α-promoter to achieve substantial overexpression of wild-type GSK-3β in forebrain neurons and, therefore, in regions more relevant for AD. In this transgenic line (Tet/GSK-3β mice), GSK-3β overexpression was restricted to certain cortical neurons and hippocampal neurons. It was in the hippocampal region where a 30% increase in GSK-3β activity was observed by enzymatic assay on tissue homogenate [24]. Hippocampal overexpression of GSK-3β resulted in an increase in the phosphorylation of Tau in Tet/GSK3β animals, as detected with antibodies raised against the phosphorylated tau modiﬁed in AD, like PHF-1/AD2 (Fig. 11.1B). This hyperphosphorylated Tau was found in the somatodendritic compartment, not because of an increase in the total amount of Tau protein but exclusively because of the hyperphosphorylation of Tau by GSK-3β. Despite a substantial increase in hyperphosphorylated Tau, the aberrant Tau aggregation found in AD was not observed in these GSK-3β transgenic mice. In this model, β-catenin, another GSK-3 substrate relevant to AD [25] was also analyzed. GSK-3 is a key enzyme involved in β-catenin stabilization and nuclear translocation [26]. Increased GSK-3β in Tet/GSK-3β mice was seen effective in modifying βcatenin in neurons of the CNS in vivo, since nuclear β-catenin was reduced by nearly 75% in hippocampal granular cells [23]. Considering that the genes transactivated by β-catenin are poorly characterized, these mice may serve as a good tool to identify such genes. Tet/GSK-3β mice also demonstrate neuronal stress and neuronal death as revealed the presence of reactive glia, TUNEL labeling, and cleaved caspase-3 staining. These data are in agreement with the known role of GSK-3β in the survival pathway, as well as supporting the neuroprotective effect of lithium, a relatively speciﬁc GSK-3β inhibitor [27]. The hyperphosphorylation of hippocampal Tau in transgenic Tet/GSK-3β mice, despite the lack of ﬁ lament formation, results in a behavioral impairment that can be measured in the Morris water maze test [24]. Animal models generated by using the tetracycline-regulated system make it possible to explore any potential reversal of their phenotype [28]. Thus the Tet/GSK-3β should be a good tool to test the neuroprotective effect of forthcoming GSK-3β speciﬁc inhibitors. The efﬁcacy of such inhibitors could be compared with the effect of silencing transgene expression by the administration of tetracycline to the Tet/GSK-3β mice. The tetracycline-regulated system also gives us the opportunity to overexpress the GSK-3β transgene in other regions than the CNS, using another promoter for the tetracycline activator. This would provide the great oppor-

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tTA TetO

BitetO b-Gal/GSK-3b

B

Wild-type

Tet/GSK-3b

Figure 11.1 (A) Tet/GSK-3β mice are generated by crossing mice expressing tTA under control of the CamKIIα promoter (tTA) with mice that have incorporated the BitetO construct in their genome (TetO). The double transgenic progeny (Tet/GSK3β) are expected to express GSK-3β constitutively in the brain unless tetracycline or analogues are given orally, thus preventing transactivation by tTA. (B) Hyperphosphorylation and somatodendritic localization of Tau in Tet/GSK-3β mice. PHF-1 immunohistochemistry in the dentate gyrus of wild-type or Tet/GSK-3β mice. The scale bar corresponds to 100 μm.

tunity to study the implications of a deregulation of GSK-3β in other cell types, and other tissues, and to generate animal models for other diseases. The identiﬁcation of the different mutations in the tau gene that cause FTDP-17 make clear that Tau modiﬁcations are sufﬁcient to cause neurodegeneration [29–31]. To see if the co-expression of FTDP-17 mutated tau and

208

ANIMAL MODELS WITH MODIFIED EXPRESSION

GSK-3β can amplify the phenotype observed in previously generated transgenic mice that express Tau with a triple FTDP-17 mutation (VLW mice) [32], we cross-bred in our laboratory Tet/GSK-3β and VLW mice to create triple transgenic mice, Tet/GSK-3β/VLW mice [33]. In the VLW single transgenic mice, which express the longest human Tau isoform with three FTDP-17 point mutations (G272V, P301L and R406W, hTauVLW) under the control of the modiﬁed thy1 mouse promoter, thin ﬁ laments with a width of nearly 5 nm can be observed. The triple transgenic Tet/GSK-3β/VLW mice showed an increase in Tau phosphorylation in the hippocampus, the area where the two transgenes, GSK-3β and hTauVLW, were co-expressed and where GSK-3 activity was increased. In addition hyperphosphorylated Tau with an aberrant high molecular weight was present in these triple transgenic mice but not in the single transgenic VLW mice or the Tet/GSK-3β mice. Similarly thioﬂavine-S positive neurons were only observed in the hippocampus of Tet/GSK-3β/VLW mice and ﬁ laments with a width wider than 10 nm (a width similar to that found in tauopathies) could be puriﬁed only from Tet/GSK-3β/VLW mice. One surprising observation in the triple transgenic Tet/GSK-3β/VLW mice was that the antibody 12E8 (which recognizes a phosphorylation-site that is not phosphorylated by GSK-3) correlates with the presence of a band of 64 kDa. Furthermore the thioﬂavine-S positive neurons were as well 12E8 immunostained. This is in good agreement with the fact that the 12E8 labeling of AD brains predominates in tangles when compared with other phosphoepitopes [34]. Recently it was described that the Drosophila PAR-1 kinase, and likely its mammalian homologue MARK, phosphorylated Tau at the 12E8 epitope, generating a priming residue to create a GSK-3 consensus sequence [35]. Also, neurodegeneration was found in the dentate gyrus of the Tet/GSK-3β mice [23]. Interestingly the resulting atrophy of the dentate gyrus is accelerated by the overexpression of the mutated Tau protein [33]. In conclusion, Tet/GSK-3β/VLW mice are a good animal model to study late stages of AD, since they show thioﬂavine-S positive neurons in areas affected in AD patients. In this regard it would be interesting to investigate if the accumulations formed of ﬁ lamentous Tau are reversible or if there is no return (as suggested by Santa Cruz et al. [36]) and whether neurodegeneration can be reverted. This animal model can also be very useful to test inhibitors of GSK-3β, since it recapitulates characteristics of AD in late stages like the formation of aggregates of ﬁ lamentous Tau and a severe atrophy of the dentate gyrus. In summary, the four transgenic mouse lines that overexpress GSK-3β in the brain show an increase in Tau phosphorylation (Table 11.1). Unlike the models generated by Brownlees et al., Spittaels et al., and Li et al. that do not show either an overt phenotype or evidence of neuronal degeneration, the model generated by Lucas et al. shows spatial learning deﬁcits and hippocampal neurodegeneration. Among the many possible explanations for this discrepancy are constitutive versus conditional expression, the promoters

β

β

PDGF

CaMKII-α

α-Actin

Li et al. [22]

Lucas et al. [23] Hernández et al. [24] Engel et al. [33]

Pearce et al. [39]

β(S9A)

β(S9A)

Thy1

Spittaels et al. [21] Spittaels et al. [18]

Increased tau phosphorylation at AT8 epitope without detectable GSK-3β overexpression in CNS Increased Tau phosphorylation, decreased brain volume and rescue of axonopathy in CNS of human 4-repeat Tau transgenic mice Increased Tau phosphorylation at AT8 epitope in forebrain Increased GSK-3 activity, Tau phosphorylation, astrocytosis, neurodegeneration, and decreased levels of nuclear β-catenin in forebrain; spatial learning deﬁcit in Morris Water Maze; formation of mature Tau ﬁ laments and Tau aggregates in 4-repeat mutated Tau mice Increased GSK-3β protein levels in skeletal muscle; reduction of glycogen levels and IRS-1 protein levels and increase in body weight

β and β(S9A)

NF-L MSV

Phenotype

Isoform

Brownlees et al. [16]

Promoter

Summary of transgenic mouse lines with modiﬁed GSK-3 expression

GSK-3 overexpressing mice

Reference

A.

TABLE 11.1

MOUSE MODELS THAT OVEREXPRESS GSK-3

209

Continued

KO (suppression) Heterozygote KO

FRB* loss of function allele Knock-in (point mutation) βS9 and αS21

β

β

β α and β

Hoeﬂ ich et al. [40]

Beaulieu et al. [43] O’Brien et al. [44]

Stankunas et al. [46] McManus et al. [47]

Modiﬁcation

Isoform

Reference

B. GSK-3 modiﬁcation by homologous recombination

TABLE 11.1

Embryonic lethality through massive TNF-α-induced hepatocyte apoptosis Inactivation of Akt and activation of GSK-3 through increased dopamine neurotransmission by amphetamine administration or lack of dopamine transporter; mimic of lithium treated mice in behavioral tests. Increase of β- catenin Embryonic and early postnatal lethality Knock-in mice viable and not diabetic; GSK-3β responsible for activation of muscle glycogen synthase

Phenotype

210 ANIMAL MODELS WITH MODIFIED EXPRESSION

MOUSE MODELS WITH REDUCED GSK-3 ACTIVITY

211

used, genetic background, wild-type versus constitutively active form of GSK3β (although, intriguingly, it has been recently described that Ser9 phosphorylation is necessary for Tau phosphorylation) [37], among others. AD and related tauopathies are not the only diseases that have been investigated by overexpressing GSK-3. Recent studies have demonstrated that activity and protein levels of both GSK-3α and GSK-3β are elevated in the skeletal muscle of humans suffering type 2 diabetes [38]. To investigate whether these differences might contribute to insulin resistance and glucose intolerance in type 2 diabetes, Pearce et al. have generated transgenic mice that overexpressed human GSK-3β in skeletal muscle under the transcriptional control of the human alpha-actin promoter [39]. There was a ﬁve- to seven-fold increase in skeletal muscle GSK-3β protein levels. Accordingly, glycogen synthase activation, glycogen levels and insulin receptor substrate-1 (IRS-1) protein levels were signiﬁcantly reduced in both male and female mice. The transgenic mice showed an increase in body weight (ca. 20%) because of an increase in fat mass. Male but not female GSK-3β transgenic mice had signiﬁcantly raised plasma insulin levels, and by 24 weeks of age they became glucose intolerant. The authors concluded that the overexpression of GSK-3β in skeletal muscle could lead to an impaired glucose tolerance despite raised insulin levels, consistent with the possibility that elevated levels of GSK-3β in type 2 diabetes are partly responsible for insulin resistance. 11.3

MOUSE MODELS WITH REDUCED GSK-3 ACTIVITY

Another strategy to investigate the implications of GSK-3 dysregulation is to suppress its expression. The ﬁ rst study done in a mouse model was by Hoeﬂich et al. [40]. In this study the GSK-3β gene has been disrupted by targeted deletion. These investigators showed that GSK-3β is absolutely essential for survival. The generated GSK-3β-knockout mice developed normally to midgestation but died around day 14 following massive tumor necrosis factor-α (TNF-α)-induced hepatocyte apoptosis, which could be prevented by the injection of antibodies that block the function of TNF-α. Nuclear factor κB (NF-κB) activation is known to counteract TNF-α-induced apoptotic signaling to promote survival by turning on a set of anti-apoptotic genes [41]. The intriguing ﬁ nding made by Hoeﬂ ich et al. is that GSK-3β is required for the NF-κB-mediated survival response. This study therefore demonstrates for the ﬁ rst time an isoform-speciﬁc GSK-3 function, since GSK-3α was unable to compensate for the loss of GSK-3β. Although the GSK-3β knockout mice died during midgestation, heterozygous mice were viable and appeared morphologically normal. Mouse embryonic ﬁbroblasts derived from the GSK-3β heterozygous knockout mice expressed reduced levels of GSK-3β protein without a compensatory increase in GSK-3α protein levels, indicating a partial loss of function in GSK-3β heterozygous. The authors also observed a reduced GSK-3β activity in the brains of the heterozygous mice [40].

212

ANIMAL MODELS WITH MODIFIED EXPRESSION

These GSK-3β heterozygous knockout mice were used by Beaulieu et al. and O’Brian et al. in two recent studies. In the ﬁ rst study Beaulieu et al. demonstrated in dopamine transporter knockout mice that this monoaminergic neurotransmitter implicated in multiple brain disorders such as Parkinson’s disease, schizophrenia, or attention deﬁcit hyperactivity disorder [42] can exert its behavioral effects by acting on a lithium-sensitive signaling cascade involving Akt/PKB and GSK-3 [43]. In this study increased dopamine neurotransmission arising either from administration of amphetamine or from the lack of dopamine transporter resulted in inactivation of Akt and concomitant activation of GSK-3α and GSK-3β. These biochemical changes were effectively reversed by the administration of the GSK-3 inhibitor lithium. The GSK-3β heterozygous knockout mice reproduced the effect of lithium in behavioral tests, thus establishing this cascade as an important mediator of dopamine action in vivo. O’Brien et al. compared the behavioral effects of chronic lithium treatment on mice with the behavioral phenotype of the GSK-3β heterozygous knockout mice [44]. In this study the authors observed that lithium-treated mice spent less time immobile in the forced swimming test, test widely used as a predictor of antidepressant efﬁcacy. The same result was obtained when they used the GSK-3β heterozygous knockout mice. In the exploratory test both lithiumtreated and GSK-3β heterozygous knockout mice acted in the same way with less exploratory activity as compared to wild-type mice. Molecular targets of GSK-3 dependent signaling, such as β-catenin, were also affected similarly by lithium and GSK-3β haploinsufﬁcience with a substantial increase of nearly 30%. These behavioral and molecular correlations strongly support the hypothesis that GSK-3 is an important target for the behavioral effects of lithium. The study of a long-term genetic suppression of the GSK-3 activity was difﬁcult to achieve because of the embryonic lethality reported by Hoeﬂ ich et al. [40]. As an alternative approach, we generated in our laboratory transgenic mice that express GSK-3β with the K85R point mutation [45] under the transcriptional control of the same conditional tetracyclineregulated system used before in the Tet/GSK-3β mice [23]. The K85R mutation results in a dominant negative form of GSK-3β. By crossing these tet/DN-GSK-3 mice with distinct transgenic mice for the tetracyclineregulated system, we expect to be able to explore the effect of a long-term suppression of GSK-3β in different tissues. Furthermore, by shutting down the transgene expression, it would be possible to study if the effects of longterm suppression are reversible. To suppress the activity of GSK-3, Stankunas et al. followed another strategy. In this study the authors generated a mouse line [46] in which proteins encoded by the endogenous GSK-3β loci are modiﬁed with sequence encoding an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3β, thus producing a severe loss-of-function allele. In the presence of a nontoxic, cell-permeable small-molecule rapamycin-derivative (C20-MaRap), GSK-

MOUSE MODELS WITH MODIFIED FORMS OF GSK-3

213

3βFRB* binds to the ubiquitously expressed FKBP12 protein, and this interaction stabilizes GSK-3βFRB* and restores both protein levels and activity. GSK-3β+/FRB* mice are viable and fertile but most GSK-3βFRB*/FRB* occasional pubs died immediately following birth. In this regard GSK-3β knockdown by loss of function allele seems to result in a phenotype less severe than that of GSK-3 KO mice. However, Stankunas et al. mention that in their laboratory the phenotype of GSK-3βFRB*/FRB* mice is indistinguishable from that of GSK-3βKO/KO mice and GSK-3βKO/FRB* mice on outbred backgrounds. To take advantage of the conditional approach used to knockdown GSK-3β in GSK3βFRB*/FRB* mice, the authors explored the possibility to deliver C20-MaRap to embryos. For this, E10.0 pregnant heterozygous GSK-3βFRB* mice (crossed to heterozygous males) were injected with 200 mg/kg/day of 20-MaRap. Although GSK-3βFRB* protein levels were partially restored in E11.5 embryos, the authors concluded that new and more efﬁcient rapamycine derivatives will be required for effective stabilization in vivo.

11.4

MOUSE MODELS WITH MODIFIED FORMS OF GSK-3

The third approach used to study the dysregulation of GSK-3 is the generation of knock-in mice with a modiﬁed GSK-3 transgene. Recently McManus et al. have generated GSK-3 homozygous knock-in mice in which the protein kinase B phosphorylation sites on GSK-3α (Ser21) and GSK-3β (Ser9) were changed to a nonphosphorylable Ala residue [47] to study the role played by the inactivation of GSK-3 through these phosphorylation events in insulin and Wnt signaling pathways. These knock-in mice were viable and were not diabetic, despite insulin being unable to stimulate glycogen synthase in muscle. This, according to the authors, can be explained by hepatic glycogen synthase not being perturbed in the knock-in mice, thus enabling the liver to compensate for the failure of insulin to stimulate glycogen synthase in muscle. In this study the authors showed that inactivation of GSK-3β rather than GSK-3α is the mayor route by which insulin activates muscle glycogen synthase. Moreover they pointed out that this major role of GSK-3β in skeletal muscle can be explained by the four-fold higher expression of GSK-3β compared to GSK-3α in murine skeletal muscle. The same ﬁ nding in human skeletal muscle strengthens the idea that GSK-3β may also control human glycogen synthase activity in response to insulin. They also demonstrated in these GSK-3 knock-in mice neither the activation of muscle glycogen synthase by contraction, nor the stimulation of muscle glucose uptake by insulin, nor the activation of hepatic glycogen synthase by glucose require GSK-3 phosphorylation on Ser21/Ser9. In embryonic stem cells of the homozygous GSK-3 knock-in mice the Wnt3a ligand of the Wnt signaling pathway induced normal inactivation of GSK-3, leading to stabilization of β-catenin and induction of β-catenindependent transcription. This Wnt-mediated inactivation of GSK-3 being

214

ANIMAL MODELS WITH MODIFIED EXPRESSION

independent of phosphorylation of GSK-3α at Ser21 and GSK-3β at Ser9 is in good agreement with the observation that the GSK-3 knock-in mice develop normally.

11.5

NONMURINE ANIMAL MODELS

Besides the mouse, other organisms have been used to explore the effects of a deregulation of GSK-3. Most efforts have been made in Drosophila melanogaster, but there have been also some reports on research made in the zebraﬁsh. The fruit ﬂy Drosophila is providing a useful model for transgenic studies of neurodegeneration. Drosophila is extensively characterized, relatively quick to manipulate, and can be employed in forward genetic screens to identify modiﬁer genes of a pathogenic phenotype [48]. In this animal model Jackson et al. showed, that Tau overexpression, previously shown to be neurontoxic [49], in combination with phosphorylation by the Drosophila GSK-3β orthologue shaggy/zeste, exacerbated neurodegeneration induced by Tau overexpression alone. Furthermore the co-expression of Tau and GSK-3β led to neuroﬁbrillary pathology in the ﬂy with the formation of abnormal Tau ﬁ laments [50]. These results are in good agreement with the results obtained from the Tet/GSK-3β/VLW mice [33], strengthening the importance of GSK3β in this process. Jackson et al. also demonstrated in this model that the manipulation of components of the Wnt signaling pathway like β-catenin/ armadillo modulates neurodegeneration induced by Tau pathology. In regard of these results the authors suggest that the disease could occur independently of Tau aggregate formation, with Wnt signaling components downstream of GSK-3β modulating Tau-induced neurodegeneration. In a more recent study Mudher et al. investigated the consequences of Tau and GSK-3β overexpression on axonal transport in Drosophila [51]. Here they show that Tau overexpression disrupts axonal transport, causing vesicle aggregation, and this was associated with loss of locomotor functions, without evidence of cell death. The co-expression of a constitutively active form of GSK-3β/shaggy enhanced this phenotype, and the administration of GSK-3β inhibitors reversed both the axonal transport- and locomotor-phenotype. The authors suggest that the pathological effects of Tau were phosphorylation dependent. Despite the well-characterized biology of the zebraﬁsh and this model system’s usefulness in elucidating the pathogenic basis of several human diseases, only one study has attempted to use this model to overexpress GSK-3. In this case Tokuoka et al. studied the molecular basis of synapse formation in the CNS [52]. The authors used a zebraﬁsh model with enhanced green ﬂuorescent protein (EGFP) expression to visualize the development of the entire retinal ganglion cell (RGC) axon projection to the tectum. Here the authors observed that the expression of a dominant-negative form of zebraﬁsh

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GSK-3β suppressed the arborization ﬁeld of RGC axon terminals in the tectum. They also found that the suppression of GSK-3β activity increased the size of vesicle-associated membrane protein 2-EGFP puncta in RGC axon terminals at the early stage of innervation to the tectum. They suggested that GSK-3β regulates the arborization ﬁeld and maturation of RGC axon terminals, thus strengthening the idea of GSK-3β playing an important role in development. 11.6

CONCLUDING REMARKS

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51. Mudher A., Shepherd D., Newman T.A., Mildren P., Jukes J.P., Squire A., Mears A., Drummond J.A., Berg S., MacKay D., Asuni A.A., Bhat R., Lovestone S. (2004). GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila. Mol. Psychiat., 9, 522–530. 52. Tokuoka H., Yoshida T., Matsuda N., Mishina M. (2002). Regulation by glycogen synthase kinase-3beta of the arborization ﬁeld and maturation of retinotectal projection in zebraﬁsh. J. Neurosci., 22, 10324–10332.

PART III GSK-3 INHIBITORS: DEVELOPMENT AND THERAPEUTIC POTENTIAL

12 LITHIUM, THE SEMINAL GSK-3 INHIBITOR Richard S. Jope Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham

Contents 12.1 Introduction 12.2 Regulation of GSK-3 12.3 Lithium Directly and Selectively Inhibits GSK-3 12.4 Lithium Promotes Inhibitory Serine-Phosphorylation of GSK-3 12.5 Outcomes of Lithium’s Inhibition of GSK-3 12.6 Therapeutic Applications: Lithium or Other Inhibitors of GSK-3? 12.7 Concluding Remarks

12.1

INTRODUCTION

The hypothesis that glycogen synthase kinase 3 (GSK-3) is a valid drug target was actually tested before it was conceptualized, since lithium was used therapeutically for nearly 50 years before it was known to inhibit GSK-3. Patients with bipolar mood disorder, previously called manic-depression, have been given lithium since 1949 [1], but not until 1996 was lithium discovered to be an inhibitor of GSK-3 [2]. This discovery greatly accelerated studies of the cellular processes regulated by GSK-3, which have proved to be much more extensive than previously imagined. Studies employing lithium Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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as a selective inhibitor of GSK-3 also provided important insights in the recent revelations that GSK-3 can inﬂuence several prevalent diseases, such as diabetes, cancer, and neurodegenerative diseases, in addition to psychiatric diseases. These discoveries using lithium to inhibit GSK-3 led many investigators to search for additional inhibitors of GSK-3, which has clearly been a successful endeavor as shown by the present book. This chapter reviews the discovery, characterization, and cellular effects of lithium, the ﬁ rst pharmacological and therapeutic inhibitor of GSK-3.

12.2

REGULATION OF GSK-3

Endogenous physiological mechanisms are important factors that inﬂuence the mechanisms, magnitude, and ramiﬁcations of the inhibitory effects of lithium on GSK-3. In order to fully understand the actions of lithium, it is important to be familiar with these endogenous regulatory mechanisms. These mechanisms have been categorized into four classes: phosphorylation, protein-protein interactions, localization, and substrate preparedness [3]. Phosphorylation is a major physiological mechanism regulating GSK-3. The activity of GSK-3 is inhibited by phosphorylation of an N-terminal serine in each of the two isoforms of GSK-3, which comprises serine 9 in the 47 kDa GSK-3β and serine-21 in the slightly larger 51 kDa GSK-3α. This inhibition is caused by the phospho-serine-9/21 forming an intramolecular association with a region that otherwise binds to substrates; thus the phospho-serine 9/21 acts as a pseudosubstrate that blocks the access of substrates to GSK-3 [4,5]. The inhibitory serine-phosphorylation of GSK-3 can be catalyzed by several kinases, the most predominant apparently being Akt (also called protein kinase B), but others such as protein kinase A (cyclic AMP-dependent protein kinase) and protein kinase C also can phosphorylate the regulatory serine [6]. Tyrosine phosphorylation of GSK-3 (tyrosine-216 in GSK-3β and tyrosine-279 in GSK-3α) also may contribute to regulation of GSK-3 by facilitating its activity, but because of evidence that this may be catalyzed by constitutive autophosphorylation the regulatory impact of tyrosine phosphorylation of GSK-3 in vivo currently is not clear [7]. GSK-3 is known to phosphorylate more than 40 substrates [3], so further regulation is needed to direct or inhibit its phosphorylation of speciﬁc substrates. One mechanism for this is regulation by GSK-3 binding proteins that target the action of GSK-3 toward speciﬁc substrates. The best known of these are GSK-3 binding proteins in the Wnt signaling pathway. In this pathway the scaffold protein, Axin, and other associated proteins bind GSK-3 to direct its actions to phosphorylate a speciﬁc substrate, β-catenin. It appears that GSK-3 bound to Axin only phosphorylates proteins within the Axin multiprotein complex. Thus regulation of the proteins in this complex modulates only the activity of GSK-3 directed toward substrates within the complex. Similar regulation by other GSK-3 binding proteins is likely a common mechanism

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outside of the Wnt pathway to selectively direct and regulate the activity of GSK-3 toward other substrates [3]. Thus GSK-3 binding proteins are critical for regulating the actions of speciﬁc pools of GSK-3 to control its phosphorylation of individual target substrates. The action of GSK-3 also is controlled by the regulation of its subcellular localization. For example, nuclear levels of GSK-3 are dynamically modulated [8], and this affects its ability to regulate nuclear substrates [9], such as a relatively large number of transcription factors that are phosphorylated by GSK-3 [6]. GSK-3 is also located within mitochondria, but little is known about how this pool of GSK-3 is regulated and what targets for phosphorylation by GSK3 exist in mitochondria [10,11]. Spatially restricted regulation of GSK-3 also occurs in growth cones at the ends of neuronal processes [12], and it seems likely that other subcellular pools of GSK-3 are restrictively regulated [3]. Finally, the actions of GSK-3 are usually regulated by the phosphorylation state of its substrate. This is because most of the substrates of GSK-3 must be “primed,” meaning prephosphorylated, at a residue 4-amino acids C-terminal to the GSK-3 phosphorylation site. This necessitates temporal coordination of the activity of the priming kinase along with GSK-3 activity for the latter to phosphorylate the primed substrate. These four regulatory mechanisms, phosphorylation, protein complexes, subcellular localization, and substrate phosphorylation, must be spatially and temporally orchestrated to provide substrate-speciﬁc regulation of the phosphorylation by GSK-3 [3].

12.3

LITHIUM DIRECTLY AND SELECTIVELY INHIBITS GSK-3

A very large number of biochemical effects of lithium have been reported, but few direct targets are known [13]. The ﬁ rst enzyme known to be directly inhibited by lithium is phosphoglucomutase, which regulates the interconversion of glucose-1-phosphate and glucose-6-phosphate [14]. Perhaps the most intensively studied direct target of lithium is inositol monophosphatase, an enzyme in the phosphoinositide second messenger pathway [15,16]. Subsequently other inositol polyphosphatases [17,18] and the related enzyme bisphosphate 3′-nucleotidase 1 [19] were found to be directly inhibited by lithium. Thus, prior to the discovery that GSK-3 is a direct target of lithium, several other enzymes were known to bind and be inhibited by lithium. Glycogen synthesis had been known for many years to be increased by lithium, but the site of action was not identiﬁed, even though investigators had suggested that lithium may inhibit a protein kinase acting on glycogen synthase [20]. However, the discovery that lithium inhibits GSK-3 was based not on studies of glycogen synthesis but on insightful observations of the effects of lithium on Xenopus laevis development [2]. These investigators observed that lithium treatment caused dorsalization similar to that caused by ectopic expression of Wnt genes in Xenopus laevis embryos, and it was known that Wnt signaling caused inhibition of GSK-3β. Experiments with

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puriﬁed GSK-3β conﬁ rmed the deduction that lithium inhibits GSK-3β, and demonstrated that lithium directly inhibits the enzyme with an IC50 of approximately 2 mM [2]. Thus the ﬁ rst known inhibitor of GSK-3 was identiﬁed as a result of basic research on development. The initial study and several subsequent reports unequivocally demonstrated that lithium is a direct and selective inhibitor of GSK-3 and also established the invaluable utility of lithium for identifying the actions of GSK3, which have turned out to be surprisingly numerous. Klein and Melton [2] demonstrated that in vitro lithium inhibited the GSK-3β-mediated phosphorylation of two of its known substrates, Tau protein and protein phosphatase inhibitor-2. They also began to verify the selectivity of lithium’s inhibitory effect on GSK-3, as they showed that lithium did not inhibit several other protein kinases [2]. Shortly thereafter Stambolic et al. [21] also reported lithium’s selective inhibitory inﬂuence on GSK-3β, and further showed that lithium was an effective inhibitor of GSK-3β in intact cells by assessing its inhibitory effect on GSK-3β-mediated phosphorylation of Tau. Although not surprising considering lithium’s well-known ability to traverse membranes through ubiquitous sodium channels, these were important results because they supported the feasibility of using lithium in intact cells and tissues to dissect signaling activities and cellular functions that are dependent on GSK3. It was also shown that lithium inhibits both isoforms of GSK-3, GSK-3β and GSK-3α [21], which emphasizes the importance of recognizing that although many effects of lithium have been attributed to inhibition of GSK3β, inhibition of either or both isoforms of GSK-3 was actually involved. The speciﬁcity and mechanism of lithium’s inhibitory effect on the two isoforms of GSK-3 have been examined in further detail. Davies et al. [22] reported an extensive study of 24 different kinases, and found that no other kinase was inhibited more than GSK-3β by lithium, although mild inhibition by lithium of casein kinase 2 and mitogen-activated protein kinase-activated protein kinase 2 was found. The concentration of magnesium was shown to inﬂuence the extent of inhibition of GSK-3 by lithium [22], and subsequently it was shown that lithium is a competitive inhibitor of GSK-3β with respect to magnesium [23]. Davies et al. also noted that the in situ inhibitory effect of lithium may be greater than that observed in the initial in vitro studies with puriﬁed GSK-3β because lithium’s potency is greater in the presence of the lower concentrations of magnesium found intracellularly compared to the higher concentrations used in the in vitro assays [23]. Another important effect of lithium is that it appears to inhibit GSK-3 throughout the cell, including GSK-3 bound in protein complexes and GSK-3 sequestered in organelles or subcompartments of cells. For example, lithium is able to inhibit GSK-3 bound in the large multiprotein complexes associated with the Wnt signaling pathway. This is evident because through its inhibition of GSK-3 in these complexes, lithium increases the cytoplasmic levels of βcatenin in the Wnt pathway that is vulnerable to enhanced degradation as a result of GSK-3-mediated phosphorylation [21]. This represents the ﬁ rst

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ﬁ nding that lithium can inhibit GSK-3 within protein complexes, a characteristic that has been conﬁ rmed with studies of GSK-3 in other protein complexes [24,25]. Further lithium can inhibit GSK-3 in the nucleus and all other cellular compartments that have been investigated [11]. Thus lithium is especially valuable because it apparently can inhibit GSK-3 even while GSK-3 is associated with other proteins, and it inhibits all cellular pools of GSK-3, properties not yet studied for some of the newer inhibitors of GSK-3.

12.4 LITHIUM PROMOTES INHIBITORY SERINE-PHOSPHORYLATION OF GSK-3 It is now known that the inhibitory effect of lithium on GSK-3 activity is achieved by two mechanisms that act in concert. First, as discussed in the previous section, there is the well-known direct inhibitory effect by lithium on GSK-3 through competition with magnesium ions for binding to GSK-3. Additionally, as was recognized more recently, lithium has a second inhibitory effect on GSK-3: lithium causes indirect inhibition of GSK-3 by increasing the inhibitory serine-phosphorylation of GSK-3 [reviewed in 26]. The initial studies of lithium’s effect on GSK-3 unequivocally demonstrated that lithium binds directly to GSK-3, which inhibits its activity. However, the relatively modest inhibition of GSK-3 that is likely to be achieved by concentrations of lithium that can be attained by in vivo administration to mammals without causing severe toxicity is only approximately 1 mM lithium, which is less than the IC50 for GSK-3 inhibition. This raised the question of whether or not this level of lithium could have signiﬁcant effects on the activity of GSK-3 in vivo. Speciﬁcally, there was speculation that the modest inhibition of GSK-3 achieved with therapeutic concentrations of lithium near 1 mM might not have a signiﬁcant effect on GSK-3 function in intact tissues, especially in the brain, since this is the presumed target organ of therapeutic lithium in bipolar mood disorder. This issue appears to have been resolved by recent ﬁ ndings showing that GSK-3β is inhibited by a second mechanism following lithium treatment, which involves increased inhibitory phosphorylation of GSK-3β on serine 9 [reviewed in 26]. Importantly, this inhibitory modiﬁcation of GSK-3β was achieved in vivo in mouse brain following chronic administration of lithium at a therapeutically relevant dose [27]. Lithium proved not only to directly inhibit the activity of GSK-3 but also to increase the inhibitory serine phosphorylation of GSK-3, providing an ampliﬁcation mechanism that allows the two mechanisms to act in concert to achieve substantial inhibition of GSK-3 with a therapeutic concentration of lithium. Therefore tolerable doses of lithium given in vivo can signiﬁcantly lower the activity of GSK-3. Because lithium has many biochemical effects, it is still not known how inhibition of GSK-3 by lithium contributes to its therapeutic actions, but there is no doubt that GSK-3 is inhibited in patients treated with lithium.

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The mechanism by which lithium treatment increases serine phosphorylation of GSK-3 remains under investigation. Currently it appears that more than one mechanism underlies this modiﬁcation, possibly in a cell-type selective manner. As summarized by Zhang et al. [28], several research groups found increases in serine phosphorylation of GSK-3α and/or GSK-3β following in vitro studies with a wide range of concentrations of lithium [27,29,33]. The ﬁ rst mechanism proposed for this effect was that lithium-activated phosphatidylinositol 3-kinase (PI3K) and/or Akt resulted in increased serine phosphorylation of GSK-3 [29]. However, this signaling pathway is not activated in all tissues in which GSK-3 phosphorylation is increased by lithium treatment [27,28,31]. Kirshenboim et al. [34] reported that Akt was not involved and that protein kinase C mediated the increased serinephosphorylation of GSK-3 caused by lithium. Indirect evidence also raised the possibility that lithium may inhibit phosphatase-mediated dephosphorylation of GSK-3 [32,35]. An alternative intriguing mechanism by which lithium increases serine phosphorylation of GSK-3 was proposed by Zhang et al. [28]. They reported that inhibition of GSK-3 by lithium reduced the activity of protein phosphatase 1 (PP1) by increasing the action of the PP1 inhibitor I-2, which led to increases in the inhibitory serine-phosphorylation of GSK-3. Thus there is no question that the direct inhibitory effect of lithium on GSK-3 is ampliﬁed by an increase in the inhibitory serine-phosphorylation of GSK-3, but the mechanism remains to be more ﬁrmly established and this modiﬁcation may occur by different mechanisms in a cell-type selective manner. Taken together, these studies show that lithium has the intriguing capacity to inhibit GSK-3 in two ways, by increasing the serine-phosphorylation of GSK-3 in addition to its direct inhibitory action. These two inhibitory actions following exposure to lithium apparently act concomitantly in vivo to achieve a cumulative inhibition of GSK-3. Finding these dual inhibitory effects answers the criticisms that the direct inhibitory effect of lithium in vivo is too small to be relevant therapeutically because the phosphorylation mechanism ampliﬁes the direct inhibitory effect of lithium on GSK-3 [27]. It is notable that the increased inhibitory serine-phosphorylation of GSK-3 is a unique property of lithium not shared by other GSK-3 inhibitors [7], a characteristic that may differentiate the in vivo effects of lithium from those of other GSK-3 inhibitors.

12.5

OUTCOMES OF LITHIUM’S INHIBITION OF GSK-3

The identiﬁcation of a speciﬁc inhibitor for any enzyme provides a valuable investigative tool. Such has been the case with the use of lithium as a selective inhibitor of GSK-3. However, lithium has other targets in addition to GSK-3. Although lithium has been reported to affect numerous cellular functions [13], only for a limited number of actions can the effects of lithium be attributed to inhibition of GSK-3 based on existing data. Discussed below are some

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examples of the actions of lithium that clearly can be attributed to inhibition of GSK-3. The discovery that lithium inhibits GSK-3 provided the basis for many investigations that have aided our understanding of the functional effects of GSK-3 as well as for studies directed toward clarifying the in vivo mechanisms of action of lithium. Several of the initial studies of the inhibition of GSK-3 by lithium focused on β-catenin. Phosphorylation of β-catenin by GSK-3 enhances the degradation of β-catenin, and several early studies found that lithium treatment inhibited this effect, which led to increased levels of βcatenin [21,36,37]. However, lithium was also reported to activate β-catenin signaling by a mechanism other than modulation of β-catenin levels through regulation of proteosomal degradation, and this has raised the intriguing possibility that lithium has additional sites of action inﬂuencing β-catenin signaling [38]. Studies of β-catenin demonstrated that lithium activates the Wnt signaling pathway, which normally involves inhibition of GSK-3, an action that may account for numerous observations of developmental effects of lithium [36,39]. In this regard it is surprising that lithium does not have greater teratogenic effects on mammalian development. In addition to effects on β-catenin, lithium’s inhibition of GSK-3 also has other effects on the Wnt signaling system. For example, since phosphorylation of Axin by GSK-3β stabilizes Axin, lithium treatment resulted in decreased levels of Axin [40]. Thus inhibition of GSK-3 by lithium has contributed to a much greater understanding of the inﬂuence of the Wnt signaling pathway on development and the regulation of gene expression controlled by this signaling system. One of the most widely studied outcomes of the inhibition of GSK-3 by lithium is its modulation of microtubules. Microtubules are polymers of tubulin proteins that form a network for maintaining cellular structures and spatial organization of the interior of cells. These microtubules also provide intracellular “tracks” along which cargo, such as organelles and large protein complexes, are transported to their proper intracellular destinations by proteins known as microtubule motors, such as kinesin. However, microtubules are not analogous to steel scaffolds; instead, they are dynamically active structures that are capable of rapidly extending (increased tubulin polymerization) or collapsing (depolymerization of microtubules). These changes occur in response to signals that regulate proteins that bind and control the stability of microtubules, called microtubule-associated proteins (MAPs). This microtubule dynamicity based on MAP function allows cells to change shape, extend, or retract processes, and to move. MAPs represent one of the most widely studied groups of proteins that are substrates of GSK-3 and therefore are subject to regulation by lithium’s inhibition of GSK-3. Among the MAP family, the most widely studied as a substrate phosphorylated by GSK-3 is the protein Tau. This GSK-3 substrate has received much attention by researchers primarily because abnormally phosphorylated Tau is the major component of neuroﬁbrillary tangles that form in the brains of patients with Alzheimer’s disease and because many site-speciﬁc phosphorylation-

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dependent Tau antibodies are available [41]. Tau can be phosphorylated at many sites, and in general phosphorylation of Tau releases it from microtubules, whereas when it is dephosphorylated it tends to bind and stabilize microtubules. Many studies have used Tau to asses the inhibitory effects of lithium on GSK-3 with consistent ﬁ ndings that lithium reduced Tau phosphorylation in a wide variety of cell systems [2,21,36] as well as in vivo [42,43]. A detailed study by Hong et al. [44] demonstrated that inhibition by lithium of GSK-3-induced Tau phosphorylation enhanced the binding of Tau to microtubules and promoted microtubule assembly. More recently it was found speciﬁcally that the phosphorylation of Thr231 in Tau by GSK-3β is a critical site in regulating the ability of Tau to bind and stabilize microtubules [45]. Thus inhibition of GSK-3 by lithium causes dephosphorylation of Tau and promotes microtubule stability, an action that is thought to stabilize cellular structure. Lithium also decreased Tau aggregation in transgenic mice expressing mutant Tau [43,46]. Tau aggregation is promoted by GSK-3-mediated phosphorylation after Tau is cleaved by caspase proteases [47]. Interestingly Tau was recently found to be phosphorylated even by serine-phosphorylated GSK-3β, normally an inhibitory modiﬁcation of GSK-3β, when Ser9-GSK-3β was bound to the scaffold protein 14-3-3 [48,49]. Similarly to Tau, GSK-3 phosphorylates and regulates the function of other proteins that bind and regulate microtubules, including MAP1B [50,51], CRMP2 [52,53], CLASP [54], and APC [55,56]. Additionally the microtubule-binding molecular motor kinesin is phosphorylated by GSK-3, which inhibits kinesin-mediated anterograde transport of cellular cargo [57]. As a result of regulating the phosphorylation of these multiple microtubule-associated protein substrates of GSK-3, lithium can have a broad inﬂuence on microtubule dynamics through its inhibitory effect on GSK-3. Thus GSK-3 and its inhibition by lithium regulate many aspects of the spatial organization of cells by regulating the functions of these microtubule-binding proteins. In addition to regulating Tau phosphorylation and aggregation, lithium’s inhibition of GSK-3 has been linked to the regulation of other pathologies of Alzheimer’s disease, as previously reviewed in detail [3,6]. GSK-3 promotes, and lithium inhibits, neurotoxicity induced by the Alzheimer’s disease related toxin, amyloid β protein (Aβ), the primary component of amyloid plaques found in Alzheimer’s disease [56,58]. More recently it was found that GSK-3 increases, and lithium decreases, the production of Aβ [63–65]. Particularly interesting was the ﬁ nding that in vivo lithium treatment reduced the aggregation of insoluble Aβ in a mouse model of Alzheimer’s disease [64]. Thus, through its inhibition of GSK-3 lithium has multiple modulatory inﬂuences on Aβ, a peptide that is central to the neuropathology of Alzheimer’s disease and is able to reduce Aβ production, toxicity, and aggregation. Lithium’s inhibition of GSK-3 also overlaps with the actions of drugs used therapeutically in Alzheimer’s disease. These include two classes of drugs, cholinergic agonists and antagonists of the N-methyl-d-aspartate glutamatergic receptor, and both of these recently were found to inhibit GSK-3 in mouse brain in

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vivo, suggesting that inhibition of GSK-3 may contribute to their therapeutic actions [66]. As discussed below, lithium’s inhibition of GSK-3 provides protection from the deleterious consequences of a number of neurotoxic conditions that have been linked to the pathological mechanisms in Alzheimer’s disease, such as stress of the endoplasmic reticulum that causes accumulation of misfolded proteins [32,61,67], oxidative stress [68], inﬂammation [69], and impaired mitochondrial function [70], as well as Aβ-induced neurotoxicity. Taken together, the use of lithium to inhibit GSK-3 has provided a wealth of information in a wide variety of studies directed toward understanding the neuropathological processes active in Alzheimer’s disease that involve GSK-3. Identiﬁcation of lithium as a selective inhibitor of GSK-3 allowed the coalescence of two major ﬁelds of research, studies of the anti-apoptotic actions of lithium and of the pro-apoptotic effects of GSK-3. The ﬁ rst of these to emerge were the protective effects of lithium that were documented in several reports between 1993 and 1997, as previously reviewed [6,71]. Research on this topic greatly accelerated between 1998 and the present, during which time there has been a tremendous increase in the number of investigations demonstrating that lithium provides signiﬁcant protection to cells from potentially lethal insults. This increased attention in a large part was due to studies from Chuang and his colleagues who documented neuroprotective actions of lithium in several neurotoxic conditions [72]. Especially interesting was the ﬁ nding of a large in vivo neuroprotection provided by lithium from ischemiainduced neuronal loss [73], which subsequently was conﬁ rmed and extended in several reports [30,74,75]. There is now an extensive literature showing that lithium provides protection from a wide variety of toxic conditions, as recently reviewed [26]. This diversity indicated that lithium affects a fundamental cell survival mechanism that participates in inﬂuencing cellular responses to diverse stimuli. Although the diversity and strength of lithium’s neuroprotectant effects are well documented, less information is available about what target might encompass the protective mechanism of action of lithium. We hypothesized that neuroprotection by lithium is, at least in part, due to inhibition of GSK-3 [76]. To address this hypothesis, we established apoptotic model systems in which a particular component of apoptosis was deﬁ nitively attributable to GSK-3β. This was attained using human neuroblastoma SHSY5Y cells that overexpress GSK-3β by three- to four-fold above the endogenous level. In these cells the basal level of apoptotic cells was unaltered, but apoptosis induced by staurosporine or by heat shock was enhanced by overexpressed GSK-3β, and pretreatment with lithium blocked the GSK-3βmediated enhancement of apoptosis [71,76]. Thus a speciﬁc component of apoptosis was attributable to GSK-3β, and this component was blocked by lithium. These experiments provide a proof of principle, that lithium’s inhibition of GSK-3 can provide protection from apoptotic insults. Taken in conjunction with the increasing evidence that endogenous GSK-3 contributes to, and is necessary for, apoptosis signaling cascades induced by a broad spectrum of

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proapoptotic agents, we proposed that a key neuroprotective mechanism of action of lithium derives from its inhibition of GSK-3, a proposal that is now widely accepted as a common mechanism underlying lithium’s protective capabilities [3]. Coincident with the increasing number of reports of lithium’s protective capabilities, there was a growing literature documenting that GSK-3 promotes apoptosis. This was ﬁ rst shown in studies of the neurotoxicity caused by the Alzheimer’s disease related Aβ [58]. However, studies of the pro-apoptotic actions of GSK-3 were accelerated by a report in 1998, showing that overexpression of GSK-3 induced apoptosis [77]. Since then activation of GSK-3 has been shown to promote apoptosis in a remarkably wide variety of conditions, such as trophic factor withdrawal, phosphatidylinositol 3-kinase (PI3K) inhibition, and toxicity induced by Aβ, chemotherapeutic agents, ceramide, human immunodeﬁciency virus type 1 Tat protein, platelet activating factor, heat shock, and mitochondrial toxins, as previously reviewed [3,6]. Thus these two ﬁelds, studies of lithium’s protective actions and studies showing the promotion of cell death by GSK-3 have coalesced, and it now appears that GSK-3 is a likely target for lithium’s protective effects in a number of conditions. For example, lithium protects cells from apoptosis by inhibiting GSK-3 following p53 activation due to DNA damage [24,25,78], and stress of the endoplasmic reticulum [32,61,67,79], as well as a wide variety of other apoptotic conditions. A remaining question is to identify the targets by which GSK-3 promotes apoptosis, and these targets are currently under investigation by many laboratories. So far several targets of GSK-3 that may contribute to promoting apoptosis have been identiﬁed, as recently reviewed in detail [26], but it remains to be seen if a single target of GSK-3 is globally inﬂuencing all conditions in which GSK-3 promotes apoptosis, or if different targets of GSK-3 are important in different toxic conditions. Thus identifying the site at which lithium’s inhibition of GSK-3 promotes cell survival remains a topic of great interest. Of the many proteins phosphorylated by GSK-3, the largest category of substrates are transcription factors, as reviewed previously [6], and several of these transcription factors may be targets of GSK-3 that are involved in its facilitation of apoptosis. For example, GSK-3 is inhibitory for the activation of several transcription factors that, when activated, are well-known to contribute to cell survival. Thus under different conditions, β-catenin, AP-1, HSF-1, and CREB, all targets of GSK-3, can each promote cell survival, and their actions are inhibited by GSK-3 [6]. Therefore lithium’s enhancement of the activities of these transcription factors through attenuation of the inhibitory inﬂuence of GSK-3 provides the diversity necessary for lithium to promote cell survival against a broad array of toxic conditions. In contrast to the large literature showing that lithium’s inhibition of GSK3 promotes cellular survival from many inducers of apoptosis, the opposite outcome occurs in a different form of apoptosis, known as extrinsic apoptosis. In this condition, apoptosis is induced by activation of death receptors, such

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as is stimulated by tumor necrosis factor (TNF), and this apoptosis is facilitated, rather than blocked, by inhibition of GSK-3. As early as 1989 Beyaert and colleagues had demonstrated that lithium increases TNF-induced cytotoxicity [80]. Subsequently a landmark study showed that GSK-3β-knockout mice developed normally to midgestation, but died around day 14 following massive TNF-induced hepatocyte apoptosis [81]. This ﬁ nding not only showed that GSK-3β can be absolutely necessary for cell survival in certain conditions, it also was one of the ﬁ rst important indications of isoform-speciﬁc functions of GSK-3, since GSK-3α was unable to compensate for the loss of GSK-3β. Further studies showed that GSK-3 counteracts apoptotic signaling induced by activation of other death receptors [82,83], so inhibition of GSK-3 by lithium or other means promotes apoptosis induced by the death receptor family. Thus perhaps one of the most interesting roles of lithium-mediated inhibition of GSK-3 comes from studies of apoptosis. These have revealed that lithium can either attenuate or promote apoptosis, depending on the stimulus, with many insults recruiting GSK-3 to promote intrinsic apoptosis that is attenuated by lithium, while in death receptor-induced extrinsic apoptosis inhibition of GSK-3 by lithium increases cell death.

12.6 THERAPEUTIC APPLICATIONS: LITHIUM OR OTHER INHIBITORS OF GSK-3? Looming above all of the individual actions of GSK-3 and the effects of its inhibition by lithium and other drugs is the ultimate outcome goal: Is it possible for inhibition of GSK-3 to provide therapeutic relief from human disease? Lithium has passed this test, as it is unquestionably effective in the treatment of bipolar mood disorder. Although the therapeutic mechanism of action of lithium in bipolar mood disorder is not known, it is evident that many effects of GSK-3, and thus of lithium, can inﬂuence signaling activities, the expression of numerous genes through regulatory inﬂuences on many transcription factors, and many aspects of cell architecture and structural remodeling, each of which can be a critical component of what is known as neuronal plasticity. Thus neuronal plasticity appears to be limited by GSK-3 activity and, conversely, to be bolstered by lithium’s inhibition of GSK-3 to provide the foundation of lithium’s therapeutic effect in bipolar mood disorder. That GSK-3 is a critical target of lithium in relation to psychiatric diseases is further bolstered by recent behavioral measurements in mice. O’Brien et al. [84] tested if GSK-3 inﬂuenced behavior in the forced swim test, one of the most widely used and reliable measures of antidepressant efﬁcacy in mouse models of depressive-like behavior. They found that GSK-3 had a profound inﬂuence on this depressive behavioral response, as genetic reduction of GSK-3β greatly ameliorated depressive behavior, and lithium treatment also did so to the same extent.

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The evidence of lithium’s dual inhibitory regulation of GSK-3, and the cellular and behavioral outcomes of this inhibition, indicate that this action likely contributes to lithium’s therapeutic effects in psychiatric diseases and may be applicable to other conditions, such as Alzheimer’s disease and diabetes. However, since lithium not only inhibits GSK-3 but also inhibits other enzymes, there is currently much interest in identifying other GSK-3 inhibitors with greater speciﬁcity that may replace lithium and to extend the therapeutic applications of GSK-3 inhibitors. Unfortunately, quite a few of the newer GSK-3 inhibitors signiﬁcantly inhibit other crucial kinases, such as cyclin-dependent kinases, which may represent a difﬁcult hurdle to overcome. With further development, greater speciﬁcity by newer drugs for inhibition of GSK-3 should be achievable. On the other hand, notwithstanding its effects on other enzymes, there are certain advantages to lithium. Especially advantageous is the knowledge and experience gained from over 50 years of clinical use of lithium in patients. Lithium is the only GSK-3 inhibitor that has been used in humans, tens of thousands of people have taken lithium, and people have taken lithium for many years. Thus lithium already has a huge record of use in humans. Not surprisingly, considering this large database, a signiﬁcant number of side effects of lithium administration have been recorded, as previously reviewed [85,86], and serum levels of lithium must be monitored to maintain it at a nontoxic concentration. However, on the whole many people have tolerated lithium for many years and have greatly beneﬁted from its therapeutic efﬁcacy. Additionally lithium’s pharmacological proﬁ le is advantageous in that it easily penetrates cells, it is stable, and there appears to be no development of tolerance or sensitivity with prolonged use. Furthermore lithium’s pharmacokinetics are amenable to human administration allowing maintenance of reasonably stable intracellular levels over long periods of time, and unlike other drugs no metabolic products of lithium are generated, compounds that may add to the off-target actions of newer drugs. Thus it is a formidable challenge to discover and develop a replacement for lithium as a therapeutic inhibitor of GSK-3 based on these practical considerations, and this may also be the case based on mechanisms of action, as discussed below. It is now evident that the in vivo inhibitory effect of lithium on GSK-3 is mediated by an intriguing combination of actions: direct inhibition resulting from lithium binding to GSK-3 and inhibition by increased phosphorylation of the inhibitory N-terminal serine of GSK-3. The latter is important because it ampliﬁes a modest direct inhibition caused by a therapeutically relevant concentration of lithium to result in a more substantial inhibition of the activity of GSK-3 [26]. This unique dual mechanism of action may provide speciﬁcity for the actions of GSK-3 that are functionally inhibited by lithium for two reasons. First, serinephosphorylated GSK-3 is inhibited only from phosphorylating primed, pre-phosphorylated substrates, whereas phosphorylation of unprimed

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substrates is not impeded. Thus there is functional selectivity in lithium’s inhibitory effect of GSK-3 because the serine-phosphorylation ampliﬁes inhibition of GSK-3-mediated phosphorylation of only a portion of its substrates. Second, this serine-phosphorylation-mediated inhibitory effect of lithium on GSK-3 may not access all subcellular pools of GSK-3, perhaps providing selectivity to allow lithium to only substantially inhibit certain sub-pools of GSK-3. In other words, GSK-3 within different subcompartments of cells may be inhibited to either of two extents: GSK-3 in compartments not serine-phosphorylated after lithium treatment will be only modestly inhibited, while GSK-3 will be inhibited to a greater extent in compartments in which serine-phosphorylation is increased after lithium treatment. Which subcellular pools of GSK-3 display serine-phosphorylation-mediated ampliﬁed inhibition after lithium depends on the subcellular distribution of the enzymes mediating this modiﬁcation. This could be beneﬁcial because it may limit toxicity of lithium by limiting the pools of GSK-3 that are maximally inhibited. It is possible that greater inhibition of GSK-3 by inhibitors that act globally on all pools of GSK-3 may tend to be more toxic since it is likely critical to maintain some level of activity of GSK-3. In other words, it may prove optimal to shift the balance between active and inactive GSK-3 in certain subcellular pools rather than attaining too substantial an inhibition of GSK-3 throughout the cell as may be caused by other inhibitors. Thus this dual action of lithium raises the question of whether or not other agents that directly inhibit GSK-3 will match the actions of lithium. For example, several small molecule inhibitors of GSK-3 inhibit the serine-phosphorylation of GSK-3 in addition to directly inhibiting GSK-3, so this ampliﬁcation mechanism is inactivated [7,28]. Therefore, in order to match the inhibition of GSK-3 by lithium, greater direct inhibition of GSK-3 will need to be achieved by these molecules, and this may be difﬁcult to accomplish without causing untoward side effects due to the many actions of GSK-3. Rather than merely inhibiting GSK-3, some selectivity may need to be built into new inhibitors. For example, tissue-selective inhibition of GSK-3 may prove optimal to lessen some toxic effects. Also, as more becomes known about its subcellular localization and its association in speciﬁc protein complexes, it may prove more beneﬁcial to selectively target a pool of GSK-3 participating in speciﬁc deleterious actions. Thus selective actions on subpopulations of GSK-3 may be necessary to develop a therapeutically useful inhibitor of GSK-3 to replace lithium. Considering all of the advantages and weaknesses of lithium, will it be replaced by new inhibitors of GSK-3? For new therapeutic applications directed at peripheral organs, such as diabetes, new GSK-3 inhibitors have a reasonable chance of more effectively producing larger inhibition of GSK-3 than lithium. However, as discussed above, for central actions, such as in bipolar disorder or neurodegenerative diseases, it will be challenging to attain efﬁcacy, tolerability, and safety matching lithium.

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CONCLUDING REMARKS

Lithium is the ﬁ rst known inhibitor of GSK-3, a target identiﬁed after many years of clinical use of lithium for the treatment of bipolar mood disorder. The availability of lithium for experimental use to inhibit GSK-3 allowed investigators to study biochemical and physiological effects of GSK-3 in intact cells and in animals in vivo. Inhibition of GSK-3 by lithium assisted in the identiﬁcation of many new substrates of GSK-3 that have been found in recent years, and in deﬁ ning the actions of GSK-3 on cellular structure and plasticity, metabolism, gene expression, and cell death and survival. Thus studies with lithium in conjunction with other approaches have ﬁ rmly established GSK-3 as a broadly inﬂuential enzyme involved in many signaling pathways. Because of the links that were established between GSK-3 and several prevalent diseases, which were made to a signiﬁcant degree with the use of lithium as a selective inhibitor of GSK-3, the present book demonstrates that lithium’s inhibitory effect contributed to leading many investigators to search for additional inhibitors of GSK-3. This goal has clearly been successful, and it is now time to explore the next step to test which of these GSK-3 inhibitors can be used in therapeutic applications to alleviate diseases associated with excessive GSK-3 activity.

ACKNOWLEDGMENTS I gratefully acknowledge the research and discussions contributed by members of my laboratory. Research in the author’s laboratory was supported by grants from the National Institutes of Health.

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13 INHIBITION OF GSK-3 AS THERAPEUTIC STRATEGY IN DISEASE: EFFICACY OF AR-A014418 Ratan V. Bhat, Samantha L. Budd Haeberlein, and Johanna M. Lindquist Astrazeneca R&D Södertälje, Sweden

Contents 13.1 Introduction 13.2 GSK-3 in Alzheimer’s Disease 13.3 GSK-3 and Insulin Resistance 13.4 CNS Disorders and GSK-3 Inhibitor Lithium 13.5 Speciﬁc GSK-3 Inhibitor AR-A014418 13.5.1 AR-A014418 and Tau Splicing 13.5.2 Pharmacokinetic Properties of AR-A014418 13.5.3 In vivo Efﬁcacy AR-A014418 13.5.4 AR-A014418 in Psychiatry 13.6 Therapeutic Potential of GSK-3 Inhibitors 13.7 Concluding Remarks

13.1

INTRODUCTION

Glycogen synthase kinase 3 (GSK-3) was ﬁ rst discovered as a major kinase involved in regulation of glucose metabolism. Mammalian GSK-3 exists as two closely related isoforms GSK-3α and GSK-3β [1] of which GSK-3β is Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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highly expressed in neural tissue [2]. An alternatively splicing isoform has been described (GSK-3β2) containing 13 additional residues inserted in the catalytic domain [3]. In cells, insulin (and related growth factors) result in increased protein kinase Akt/PKB activity, which inhibits GSK-3 by phosphorylating the serine9 residue of GSK-3β or serine-21 residue on GSK-3α [4]. The serine-9 phosphorylation on GSK-3β by Akt elicits a conformational change of the N-terminus of GSK-3β, which prevents prephosphorylated substrates from physically interacting with GSK-3 [5]. On the other hand, GSK-3 activity is about 200-fold higher upon phosphorylation on a tyrosine residue (Y216) in vitro [6] and is regulated by extracellular insults in neurons [7].

13.2

GSK-3 IN ALZHEIMER’S DISEASE

GSK-3 has been associated with several neuropathological mechanisms involved in Alzheimer’s disease. The postmortem diagnosis of Alzheimer’s rests on the presence of two abnormal deposits; extracellular “plaques” consisting of β-amyloid (Aβ), and intracellular neuroﬁbrillary “tangles” (NFTs). Compared to age-matched control samples, increased levels of GSK-3 have been found in postmortem analysis of brains from Alzheimer’s disease patients [8]. In addition GSK-3 has been shown to localize to pretangle neurons, dystrophic neurites, and NFTs in Alzheimer’s disease brain [8]. Neurons actively undergoing granulovascular degeneration are also immunopositive for active GSK-3β [9]. A spatial and temporal pattern of increased active GSK-3β expression coinciding with the progression of NFT and neurodegeneration have been demonstrated. Taken together, these studies provide strong evidence that the active form of GSK-3β is increased in Alzheimer’s disease brain. Recently it has been demonstrated, using non-isoform selective pharmacological inhibitors such as lithium, kenpaullones, as well as small interfering RNA against the α isoform of GSK-3, that GSK-3 inhibition per se decreases Aβ production in cells and in an animal model of amyloidosis [10]. The exact mechanism by which this occurs is unclear. However, the observation that amyloid precursor protein (APP) C-terminal fragments accumulate in the presence of these inhibitors suggests that GSK-3 may inﬂuence γ-secretase activity. γ-Secretase activity is part of a multiprotein complex that is necessary for the terminal cleavage of APP to generate the Aβ fragment. GSK-3 is not γ-secretase itself as the inhibitors failed to demonstrate accumulation of C-terminal fragments of the Notch protein, which is also a substrate for γ-secretase [10]. If the ﬁ nding that GSK-3 inhibition reduces Aβ production is consistent, GSK-3 will gain signiﬁcant importance as a drug target for Alzheimer’s disease. Tau, a microtubule-associated protein which is abundantly expressed in the brain, binds to tubulin through three or four repeat sequences in its Cterminal portion and binding promotes microtubule assembly. Abnormal

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phosphorylation of Tau accumulates as aberrant polymers known as paired helical ﬁ laments (PHFs), the chief constituent of NFT. Upon abnormal phosphorylation, Tau reduces its afﬁ nity for and dissociates from microtubules. In Alzheimer’s disease brains it has been shown to accumulate in the neuronal perikarya and processes as PHFs. It has been suggested that at the single-cell level the defects start with a modiﬁcation of Tau by phosphorylation, resulting in a destabilization of microtubules giving rise to a “pre-tangle” stage [11]; in fact PHFs are not observed in the region where microtubules are present within the neuron. After this stage the destabilization of microtubules leads to loss of dendritic microtubules and synapses, plasma membrane degeneration, and eventually cell death [12,13]. Hyperphosphorylated Tau forms ﬁlaments in vitro [14] and in neuroblastoma cells [15]. In vitro, GSK-3 phosphorylates 95% of all sites phosphorylated in PHF Tau from Alzheimer’s brain. Some of these sites can be speciﬁcally recognized by the phosphoepitope-speciﬁc antibodies AT270, AT8, AT180, and PHF1 [16], respectively. These antibodies have been shown to recognize Tau present in PHFs in the brains of Alzheimer’s disease patients. The AT-100 epitope (Thr212/Ser214) has been linked speciﬁcally to PHF-Tau from Alzheimer’s disease brain and this antibody does not appear cross react with fetal Tau protein. In order to form the AT-100 epitope, a sequential phosphorylation by PKA and subsequent phosphorylation by GSK-3 is required [17]. Persistent Tau phosphorylation could result in neuritic dystrophy. Lipophosphatidic acid treated neurons result in GSK-3 dependent persistent Tau phosphorylation followed by neurite retraction and growth cone collapse [18]. Several animal models, which exhibit persistent Tau phosphorylation, also display neuritic dystrophy. For example, mice lacking either Reelin, mammalian disabled (mDab1), or VLDLR2 and ApoER2 exhibit persistent Tau phosphorylation and have neuritic dystrophy and cytoskeletal abnormalities associated with them [19,20]. It is conceivable that persistent phosphorylation by GSK-3 results in neuritic dystrophy and subsequent cytoskeletal breakdown. In Drosophila Tau overexpression, in combination with phosphorylation by the Drosophila GSK-3 homologue Shaggy, exacerbated neurodegeneration induced by Tau overexpression alone, leading to neuroﬁbrillary pathology [21]. GSK-3 has been shown to phosphorylate and inhibit kinesinmediated motility. Fast axonal transport misregulation has been hypothesized to play a role in Alzheimer’s disease pathogenesis [22]. Fibrillar Aβ binds to and induces the clustering of the integrin receptors, leading to the activation of paxillin and focal adhesion kinases. Interestingly it was observed that active GSK-3 associates with focal adhesion proteins [7], and it has been hypothesized that GSK-3 could mediate neuritic dystrophy via these interactions [23]. Abnormal increase in GSK-3 activity has been shown to cause neurodegeneration and interfere with synaptic plasticity [for a review, see 24, 25].

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In a conditional GSK-3 transgenic mouse overexpression of GSK-3 in the hippocampus and cortex resulted in Tau hyperphosphorylated at the PHF1 site and pre-tangle-like somatodendritic localization of Tau. Neurons with this Tau localization often had abnormal morphologies and neuronal death was also observed [26]. Thus this mouse model exhibits some of the features observed in Alzheimer’s disease and can be used as a system to test the efﬁcacy of GSK-3 inhibitors.

13.3

GSK-3 AND INSULIN RESISTANCE

GSK-3, one of the ﬁ nal regulatory proteins in glycogen production, is an attractive target for non-insulin-dependent diabetes mellitus (NIDDM). In NIDDM biopsy tissue GSK-3 activity is inversely correlated to glycogen synthase activity implicating GSK-3 as contributing to insulin resistance [27,28]. Insulin and insulin receptors are expressed in the brain, including the medial temporal regions that support the formation of memory [29]. Some NIDDM patients are also afﬂ icted with verbal and visual memory decline independent of clinically diagnosed dementia [30]. More recently evidence suggests that the insulin resistance known to underlie NIDDM may also contribute to the clinical symptoms of Alzheimer’s disease. GSK-3 intersects these two diseases, and therapeutic inhibition of GSK-3 is indicated for both.

13.4

CNS DISORDERS AND GSK-3 INHIBITOR LITHIUM

In FTDP linked to chromosome 17, the presence of some mutations in Tau correlates with the onset of the disease. In this case, it has been found that those mutations could affect to the dephosphorylation of Tau molecule which is already pre-phosphorylated [31], and this pre-phosphorylation could be due to GSK-3 [32]. Tau mutations that result in Frontotemporal dementia (FTD) consist of NFT pathology. Transgenic mouse models with these mutations (P301L, V337M, R406W) also exhibit NFT like pathology. It has been shown that kinases such as GSK-3 and possibly cdk5 activity are increased in these mouse models. Treatment of transgenic mice overexpressing mutant human tau (P301L, 4RON), with the GSK-3 inhibitor lithium has been shown to signiﬁcantly lower levels of Tau phosphorylation and signiﬁcantly reduce levels of aggregated, insoluble Tau [33]. Administration in this model of a second GSK-3 inhibitor, AR-A014418, also correlated with reduced insoluble Tau levels, supporting the idea that lithium exerts its effect through GSK-3 inhibition [33]. Following middle cerebral artery occlusion in mice, which results in cortical infarcts, an increase in GSK-3 activity was shown to coincide with cell death [7], and a reduction in infarct volume with the GSK-3 inhibitor lithium was demonstrated [34], indicating that GSK-3 inhibition may be beneﬁcial in

SPECIFIC GSK-3 INHIBITOR AR-A014418

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stroke. The observation that diverse stressors activate GSK-3 may also explain how lithium can show neuroprotection in a wide range of toxic insults. Lithium and valproic acid are mood stabilizers, and they are widely used in the chronic treatment of bipolar disorders. Lithium ions directly inhibit GSK-3 [35], while valproic acid inhibits GSK-3 activity in relevant therapeutic concentrations in human neuroblastoma cells [36]. The mechanism of action by which lithium exerts its therapeutic effects is not known, but therapeutic efﬁcacy is observed after several days of treatment. It is therefore conceivable that the acute effects on GSK-3 result in changes in gene regulation and cellular changes that could affect the neuronal plasticity over time [37,38,39]. Lithium inhibits at least four phosphomonoesterases (including inositol monophosphatase) [40], phosphoglucomutase [41,42], and GSK-3 [35,47]. However, GSK-3 is signiﬁcantly inhibited at therapeutic lithium concentrations (0.6–1.2 mM) [10,39,43]. The Ki for lithium’s inhibition of GSK-3 is approximately 1–2 mM [35]. Lithium’s inhibition of GSK-3 is thought to be by competing for magnesium [44,45] whose estimated brain levels are between 0.2 and 1.2 mM [46,47]. Chronic lithium treatment results in increased protein levels of the GSK-3 substrate, β-catenin, in rat brain [48]. If a signiﬁcant proportion of lithium’s therapeutic actions in bipolar disorder results from the inhibition of GSK-3, then GSK-3 would be an important target for bipolar disorder [49]. Recently lithium has been reported to regulate circadian rhythm cycles through inhibition of GSK-3. Deregulation of circadian rhythms (sleep-wake cycles) is observed in both bipolar disorder and unipolar depression [50]. In Drosophila, activation of GSK-3 or Shaggy advances the entry of the Timeless protein into the nucleus [51]; a direct translation in mammals would be an advancement of circadian rhythms. Lymphocytes of patients with schizophrenia have impaired GSK-3 protein levels and activity [52], and GSK-3 is reduced in the frontal cortex of postmortem schizophrenic brains [53]. Since the Wnt family of genes plays a central role in normal brain development, it is possible that during development impairment in GSK-3 may lead to abnormal neuronal development.

13.5

SPECIFIC GSK-3 INHIBITOR AR-A014418

To date, several GSK-3 inhibitors have been described and most of the observed effects are in vitro and cellular studies [reviewed in 25]. Among the numerous GSK-3 inhibitors described in the literature, only limited selectivity versus closely related kinases CDK2 and CDK5 has been demonstrated [25,54]. In a therapeutic context for CNS disorders, sufﬁcient separation between efﬁcacious brain concentrations and levels is essential and may result in side effects as a consequence of nonselective kinase inhibition. A potent small-molecule GSK-3 inhibitor belonging to the thiazole chemical class AR-A014418 has been reported by AstraZeneca [55]. AR-A014418

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inhibits GSK-3 by competing with ATP binding at a Ki of 38 nM. AR-A014418 inhibits Tau phosphorylation on a GSK-3-speciﬁc site (Ser396) in cells overexpressing human 4 repeat tau. In addition this inhibitor is highly speciﬁc for GSK-3, as it does not signiﬁcantly inhibit 26 other kinases tested, including CDK2 and CDK5. The crystal structure of GSK-3 in complex with ARA014418 (Fig. 13.1) shows that the inhibitor binds to the backbone atoms of the Val135 residue located in the hinge/linker region through three hydrogen bonds. These interactions alongside of the ATP binding pocket and a Glu-Arg salt bridge that deﬁ nes the ﬂat boundary of the pocket provide insight in the structural basis for selectivity. GSK-3 activity is responsive to diverse apoptotic insult in neuronal cells. In agreement with this, GSK-3 inhibition promotes cell survival in growth factor-deprived PC12 cells by mechanisms upstream of caspase-3 signaling [56]. Similarly GSK-3 inhibition by AR-A014418 protects N2A neuroblastoma cells against cell death induced by inhibition of the phosphatidylinositol 3-kinase/protein kinase B pathway, a pathway essential for cell survival. Furthermore AR-A014418 inhibits amyloid-induced neurodegeneration in hippocampal slices, which exempliﬁes the usefulness of this compound for studying the involvement of GSK-3 in neurodegenerative processes [55]. 13.5.1

AR-A014418 and Tau Splicing

Recently a novel role for GSK-3 was demonstrated in transcriptional processes related to alternative splicing of human Tau exon 10, providing

Figure 13.1 X-ray crystal structure of GSK-3 co-crystallized with GSK-3 inhibitor AR-A014418 showing binding within the ATP pocket. See color plates.

SPECIFIC GSK-3 INHIBITOR AR-A014418

249

evidence for pleiotrophic involvement of GSK-3 in Tau pathology [57]. Cortical neurons treated with AR-A014418 display increased proportion of the Tau 4-repeat (4R) isoform as a consequence of alternative exon 10 splicing, supporting a role for GSK-3 in alternative Tau splicing. The suggested mechanism by which GSK-3 modulates Tau splicing involves phosphorylation of serine/ arginine-rich (SR) splice factor SC35, which have been shown to co-localize with GSK-3 in nuclear speckles. By phosphorylating spliceosome components and thereby affecting the formation of the active complex, it is feasible that GSK-3 activation may result in inhibition of alternative splicing events. Even though the precise mechanism by which GSK-3 affects pre-mRNA splicing and its relevance for development of disease is not fully understood, it is possible that a shift in the ratio between Tau isoforms may result in altered microtubule plasticity (58). Altered Tau splicing is also coupled the high frequency of 5′ splice mutations located in exon 10. Importantly, altered splicing of exon 10 leading to an increase in the 4R isoform and altered ratio of 3R/4R Tau isoforms is associated with frontotemporal dementia with parkinsonism associated to chromosome 17 (FTDP-17) [59]. 13.5.2

Pharmacokinetic Properties of AR-A014418

Following a per oral dose of 1 μmol/kg of AR-A014418 in Sprague Dawley rats, the maximal concentration (Cmax) in plasma was observed to be 3.75 μM with an area under curve (AUC) of 22.4 μM*h. The half-life after per oral administration was determined to be 8.7 hours with a tmax of 0.26 hour. Following an i.v. bolus dose of 3 μmol/kg of AR-A014418, immediately followed by an infusion of 3 μmol/kg/h over 2 hours to obtain a steady state between plasma and brain levels, the brain concentration was observed to be 0.13 μM with a plasma-to-brain ratio of 0.05x. The observed, predicted and simulated values correlated extremely well using simulation with WinNonlin 4.0 modeling. The estimated concentration in brain after per oral dosing of 1 μmol/kg AR-A014418 at tmax was 0.19 μM. Doses used in most of the in vivo studies performed with AR-A014418 are at 30 μmol/kg much higher than those used in the pharmacokinetic studies and therefore brain concentrations are well above the concentrations that inhibit GSK-3 activity. 13.5.3

In vivo Efﬁcacy of AR-A014418

Animal models expressing human mutant Tau provides valuable insight regarding the therapeutic potential of GSK-3 inhibitors in tauopathies. The JNPL3 mice, expressing Tau missense mutation P301L, develop extensive NFT pathology in spinal cord and brainstem associated with progressive motor disturbances and eventually neuronal loss [60]. Treatment with 30 μmol/ kg of AR-A014418 over a period of one month signiﬁcantly reduced tauopathy in JNPL3 mice [33]. At early stages of tangle development, treatment correlated with reduced phosphorylation of soluble Tau and reduced levels of

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ﬁ lamentous Tau in brainstem, thus suggesting that GSK-3 inhibition reduce Tau phosphorylation in these mice. Administration of AR-A014418 in another model of tauopathy, the hTau mice, conﬁ rmed these ﬁ ndings. In another in vivo model selective overexpression of the wt human Tau 0N3R isoform in Drosophila motorneurons disrupts axonal transport causing vesicle aggregation and loss of locomotor function prior to neuronal death [61]. Co-expression of constitutively active GSK-3 further augments axon transport and locomotor phenotypes. AR-A014418 reverses both the axon transport and locomotor deﬁcits suggesting that this phenotype is GSK-3 dependent. Taken together, these results indicate that GSK-3 inhibition per se can attenuate the detrimental consequences of persistent Tau phosphorylation and provides additional conﬁdence that GSK-3 inhibition may be a good therapeutic strategy for reducing NFT in Alzheimer’s disease and related taoupathies. 13.5.4

AR-A014418 in Psychiatry

The therapeutic potential of GSK-3 inhibitors as an antidepressant approach has been demonstrated by the ability of AR-A014418 to affect behavioral changes in animal models. In rats, administration of AR-A014418 reduced immobility time in forced swim tests [62]. In addition the speciﬁcity of this effect is supported by studies where AR-A014418 inhibited spontaneous as well as amphetamine-induced activity. These behavioral changes are consistent with the effects of current antidepressant therapies, thus suggesting that small-molecule GSK-3 inhibitors may be useful in the treatment of bipolar disorder and depression. Thus, AR-A014418 provides an important research tool to investigate the and therapeutic potential of GSK-3 inhibition in bipolar disorder, depression, and Alzheimer’s disease as well as other neurodegenerative disorders.

13.6

THERAPEUTIC POTENTIAL OF GSK-3 INHIBITORS

The current therapeutics for Alzheimer’s disease provide marginal beneﬁt at attenuating cognitive deﬁcits by inhibiting acetylcholinesterase and increasing the levels of the neurotransmitter ACh. Unfortunately, this type of therapy does not stop the progressive neuritic dystrophy and neuronal damage, and over time these therapies become ineffective. There exists an urgent medical need to develop agents that delay or reverse the progression of Alzheimer’s disease. Until recently efforts have been focussed on preventing amyloidosis, an early event in the pathogenesis of Alzheimer’s disease. Although this is an attractive strategy, there is no clinical evidence that it will prevent neurodegeneration and NFT development. One common molecular link between amyloidosis and tau abnormalities appears to be GSK-3 [10]. In addition to Alzheimer’s disease, GSK-3 inhibition may provide therapeutic beneﬁt for multiple tauopathies that do not have the amyloidosis pathology associated with them.

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From a drug discovery standpoint, any potential side effects via acute or chronic inhibition of GSK-3 must be linked to the age of the disease population and the length of time it takes for the effect to be observed in humans. It must also be noted that GSK-3 may phosphorylate some substrates more efﬁciently. Tau is a more accessible substrate to those that are complexed with other high molecular weight proteins, and therefore there is a possibility of disconnection between efﬁcacy and any potential safety liabilities. For mild to moderate dementia patients, diagnosis is made at 65 years of age. Most patients deteriorate substantially within 4 to 5 years and death is common around 8 to 10 years after diagnosis. The majority of marketed drugs have side effects at high doses, and a balance between the doses that provide clinical beneﬁt and the dose having side effects needs to be evaluated thoroughly. Biological markers for efﬁcacy and safety may allow for appropriate dose settings in the clinic and may overcome potential liabilities.

13.7

CONCLUDING REMARKS

Abnormal elevations of GSK-3 levels and activity in the aging brain may result from several of the features associated with familial and sporadic Alzheimer’s disease such as PS-1 mutations, Aβ, oxidative stress, low phosphatase 2A activity, and excessive glutamate. Abnormal GSK-3 appears to be associated with a multitude of adverse events linked to microtubule dynamics, amyloid production, neuritic dystrophy, PHF-Tau, plasticity, and cognitive deﬁcits and neurodegeneration pointing to a key role in Alzheimer’s disease. Given the signiﬁcant role of GSK-3 in a variety of effects linked to mechanisms related to Alzheimer’s disease, GSK-3 inhibition using small-molecule inhibitors such as AR-A014418 is a testable hypothesis in the clinic.

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14 TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GLYCOGEN SYNTHASE KINASE 3 INHIBITORS Ana Castro, Mercedes Alonso, and Ana Martínez NeuroPharma Avda. de la Industria, 52, Tres Cantos, Madrid

Contents 14.1 14.2

Introduction Identiﬁcation of 2,4-Disubstituted Thiadiazolidiones 14.2.1 Synthesis of 2,4-Disubstituted Thiadiazolidinones 14.2.2 Synthesis of Heterocyclic Compounds Structurally Related to TDZDs 14.2.3 In vitro GSK-3 Activity 14.2.4 Kinases Selectivity Studies 14.2.5 Biological Effects of TDZDs 14.2.6 Structure-Activity Relationships 14.2.7 CoMFA and Mapping Studies 14.2.8 Second Generation of TDZDs 14.3 Therapeutic Potential of 2,4-Disubstituted Thiadiazolidindiones 14.3.1 Drug-like Properties 14.3.2 IGF-1 Mimetic Activity 14.3.3 Apoptosis Modulation 14.3.4 Anti-inﬂammatory Effect 14.3.5 Neuroprotectant Properties 14.4 Concluding Remarks

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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14.1

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

INTRODUCTION

The search for GSK-3 inhibitors is a very active research ﬁeld for both academic centers and pharmaceutical companies [1–5]. Until now the most frequent approach for discovering GSK-3 inhibitors has depended on exploiting screening programs that are aimed at ﬁ nding new “hits” for compounds exhibiting other pharmacological proﬁ les. This is the case for kinase inhibitors such as hymenialdisine [6], paullones [7], indirubines [8], maleimides [9], and 2,4-disubstituted thiadiazolidinones (TDZDs) [10] (Fig. 14.1). These latter derivatives were secondary products in the synthesis of potassium channel openers [11], muscarinic agonists [12], and acetylcholinesterase inhibitors [13]. However, because of the availability of X-ray crystallographic data for GSK-3β [14–18], rational drug optimization is now being applied to discover new lead compounds [19]. Within the great diversity of chemical structures with GSK-3 inhibition already found, TDZDs appeared to be the ﬁ rst ATP noncompetitive GSK-3 inhibitors [10]. These compounds are of great interest because they do not show inhibitory activity in other kinases. The potency and the selectivity of TDZD should allow their use as tool compounds in the resolution of the complex signaling pathway where GSK-3β is implicated. For this reason the regulation of GSK-3 in human platelets [20], the inhibitory phosphorylation of GSK-3 in response to lithium [21], and the involvement of GSK-3 in semaphorin 3A signaling [22] have been studied using small-molecule inhibitors such as TDZD-8 (see in Table 14.1). In addition, as we will report later in

O

H N

H2N O

O

R'

R Anilino-maleimides

R' HN R

N H

H N O

Indirubin

Paullones

Figure 14.1

Common GSK-3 inhibitors.

O

Br HN

NH O Hymenialdisine

O O

N

HN

O HN

N N R R' Bisindole-maleimides H N

H N

O R'

R N N S TDZDs

O

259

INTRODUCTION

TABLE 14.1

Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Glycogen synthase kinase 3b inhibition of thiadiazolidinones

R Et Et Et Et Et nBu cyclohex Bn Bn Bn Ph 4-BrPh 3-BrPh 2-BrPh 4-ClPh 4-FPh 4-CF 3Ph 4-MePh 4-OMePh 1-Naphthyl CH 2CO2Et Et Ph Bn Bn Et Ph Ph Ph

R′ Me Et Pr iPr cyclohe x Et Me Me Et Bn Me Me Me Me Me Me Me Me Me Me Me Et Ph Bn Et Me Et Et CO2Et

X

Y

O O O O O O O O O O O O O O O O O O O O O S S S S S O O S

O O O O O O O O O O O O O O O O O O O O O O O O O N3-Pyr NCONHEt NH NCO2Et

IC50 (μM) a 5 25 10 35 10 70 >100 2 7 10 2 3 4 6 4 4 6 5 2 3 2 20 8 10 6 10 75 65 100

a

IC 50 : 50% inhibitory concentration of GSK-3β activity (μM). Assays were performed in triplicate.

this chapter, the identiﬁcation and development of TDZDs as new drugs hold promise for the treatment of unmet diseases mediated by GSK-3 such as Alzheimer’s disease and other neurodegenerative processes in which the Tau protein is involved, as well as chronic inﬂammatory diseases and cancer.

260

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

14.2 IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLIDIONES 14.2.1

Synthesis of 2,4-Disubstituted Thiadiazolidinones

Thiadiazolidinones 1–21 were synthesized following a pathway that is based on the reactivity of N-alkyl-S-[N′-(chlorocarbonyl)amino]isothiocarbamoyl chlorides with isocyanates [23]. These heterocyclic salts are exceptionally reactive, and in the presence of moist air, it is possible, through hydrolysis, to obtain 1,2,4-thiadiazolidine-3,5-diones 1–21 as white crystalline solids after evolution of hydrogen chloride. In all the reactions assayed, the 5-oxo1,2,4-thiadiazolidin-3-thione derivatives 22–24 could be detected as minoritary products. Only when ethylisothiocyanate was used as a second reagent was the thione derivative 25 obtained as the minoritary product in low yield. Introduction of an imino moiety at the 5-position of the thiadiazolidine framework (compound 28) was achieved by the basic hydrolysis of the urea derivative 27. Compounds 27 and 29 were easily obtained following a described procedure of 5-aminophenyl-1,2,3,4-thiatriazole rearrangement with isocyanates and isothiocyanates [24].

14.2.2 Synthesis of Heterocyclic Compounds Structurally Related to TDZDs Different structural modiﬁcations were introduced in the heterocyclic ring with the aim of testing the inﬂuence of each heteroatom on biological activity [25]. As ﬁ rst approach, a common scaffold, consisting of a pentagonal ring containing carbonyl and thiocarbonyl groups in a 1,3 relative disubstitution, was explored. Different compounds such as hydantoines, dithiazolidindiones, rhodanines, maleimides, and triazoles (Fig. 14.2) were screened as GSK-3 inhibitors. The chemical modiﬁcations on the different heterocyclic systems, except for dithiazolidinedione, were achieved by the more suitable method of alkylation. Maleimides (33–38) and some rhodanines derivatives (compounds 51– 52 and 55–56) could be prepared using Mitsunobu reaction modiﬁcation [26], whereas N-alkylation of rhodanine ring, yielding 53 and 54 derivatives, occurred when we used a weak base and the corresponding alkyl halide in acetone. A previously described method [27] that combined chlorocarbonylsulphenyl chloride with different formamides was selected for the preparation of dithiazolidinedione derivatives 57–59. Different triazolidinedione (compounds 61–63) and hydantoine (compound 66) derivatives were synthesized with commercial 4-phenyl-urazole (compound 59) and hydantoine (compound 65), respectively, as the starting material in classical alkylation conditions (alkaline medium and alkyl halide) [28].

261

IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLDIONES

R N

O

R O

HN

S

Hydantoines

R N

X

R'

N

3 4 5 2 1

S

O

N S

R N

O

TDZD

S

R'

Thiazoles

R O

N

O

Dithiazolidindiones

R O

O

N

O

N N

O R'

Triazoles O

Maleidimides Figure 14.2 Schematic representation of the structural differences in the heteroaromatic rings of the different classes of compounds considered in the study.

14.2.3

In vitro GSK-3 Activity

Several compounds belonging to the TDZD family or to the previously described chemical related groups were tested against GSK-3β. This activity was determined using recombinant GSK-3β enzyme, which was incubated with ATP and GS-1 as substrate [29] in the presence and in the absence of the corresponding test compound. The results presented in Tables 14.1 through 14.4 show IC50 as the compound concentration that inhibits 50% of the enzyme activity. To investigate the mechanism of TDZD’s action on GSK-3β, several kinetic experiments were performed. The concentrations of ATP (six concentrations, from 6.5 to 100 μM) and inhibitors (two concentrations) were varied. In each case two different TDZD were used: TDZD-8 and TDZD-9. The kinase activity was measured increasing ATP up to 100 μM, the point where the enzyme saturation could be ensured. Double-reciprocal plotting of the data (Fig. 14.3), with each point representing the mean of two different experiments, suggests that TDZDs act as a noncompetitive inhibitors of ATP binding. These results provide the ﬁ rst non–ATP-competitive inhibitors of GSK-3β. To date, all the GSK-3 inhibitors described, with exception of TDZDs and thienylhalomethyl ketones [30], compete with the ATP in their mode of binding to the enzyme. This property of TDZDs inhibitors could be an important hit in their potential use in pharmacotherapy.

262

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

TABLE 14.2

GSK-3 inhibition of maleimide related compounds R Z B

A

X Y

Compound

X

Y

Z

A

B

R

30 31 32 33 34

CH CH CH CH CH

CH CH CH CH CH

N N N N N

C=O C=O C=O C=O C=O

C=O C=O C=O C=O C=O

35 36

CH CH

CH CH

N N

C=O C=O

C=O C=O

37 38 39 40 41 42 43 44 45

CH CH CH CH CH 2 CH 2 CH 2 CH 2 CH

CH CH CH CH CH 2 CH 2 CH 2 CH 2 CH

N N CH 2 O CH 2 N N N N

C=O C=O C=O C=O C=O C=O C=O C=O CH 2

C=O C=O C=O C=O C=O C=O C=O CH 2 CH 2

TABLE 14.3

GSK-3 inhibition of the rhodanine related derivatives

IC50 (μM)

H CH 3 CH 2Ph CH 2CO2Et CH 2Ph-pOCH 3 (CH 2) 2Ph (CH 2) 2Ph-pOCH 3 (CH 2) 3Ph (CH 2) 5Ph

H CH 2Ph CH 2Ph CH 2Ph

6 5 1 3 2.5 2 3 3 3 12 >100 >100 >100 >50 >100 >100

R N

O Y

X S

Compound

X

Y

R

IC50 (μM)

46 47

S S

CH 2 CH 2

100 100

48 49 50 51 52 53 54

S S O S S S O

CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2

55

S

CH 2

56

S

CH 2

H CH 3 CH 2COO H NH 2 H Bn (CH 2) 2Ph CH 2COPh Bn Bn-pOMe (CH 2) 2Php-OMe

100 100 100 25 35 50 100 65 100

263

IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLDIONES

TABLE 14.4 GSK-3 inhibition of dithiazolidindione, triazolidinediones and hydantoines derivatives R N

O X

O Y

R'

Compound

X

Y

57 58 59 60 61 62 63 64 65 66

S S S N N N N N N N

S S S N N N N N CH 2 CH 2

R''

R

R′

R″

IC50 (μM)

CH 3 Ph CH 2Ph Ph Ph Ph Ph Ph H CH 2Ph

— — — H CH 3 COPh CH 2COPh — H H

— — — H CH 3 COPh CH 2COPh — — —

100 12 12 100 100 100 100 100 100 100

TDZD-8

TDZD-9

0.4

0.35

0.35

0.30

10 μM

0.3

0.25 0.20

0.2

1/V

1/V

0.25

0.15

3.3 μM

0.15

0.10

0.1

control

0.05

0.05

0 -0.10

-0.05

0

0.05 1/[ATP]

0.1

0.15

0.2

-0.10

-0.05

0.00 0.00

0.05

0.10

0.15

1/[ATP]

Figure 14.3 Double-reciprocal plots of kinetic data determined for compounds 8 and 9. Each point is the mean of two different experiments, each analyzed in duplicate.

14.2.4

Kinases Selectivity Studies

Selectivity of protein kinase inhibition [31] is critical for pathway analysis in cellular systems as well as to avoid widespread effects in a potentially therapeutic treatment. Cdk-2 is the nearest kinase from a homology perspective

0.20

264

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

(overall 33% amino acid identity), and almost all of the reported GSK-3 inhibitors also inhibit other cdks [32]. TDZD-8 did not signiﬁcantly inhibit cdk-2 or other protein kinases such as PKA, CK-II, and PKC. Moreover, to gain an understanding of the selectivity kinases issues of this series of compounds, some selected TDZDs were run through a panel of seven related kinases (Abl-K, CAM KII, EGF-TyrK, IRK, MAPK, MEK1K, and PKp56). The inhibitory assays were performed by using a 10 μM concentration for the different inhibitors. The results in Table 14.5 show that TDZDs compounds do not, in general, have a signiﬁcant inhibitory impact on the whole set of kinases, which in turn proves the remarkable speciﬁcity of TDZDs with respect to GSK-3 [33]. In addition, a proﬁ le of neurotransmitter receptor binding assays (nonselective alpha-adrenergic type 2 (α2), human dopaminergic type 2 (D2), glutamate receptors AMPA and NMDA, nonselective muscarinic receptors (M), neuronal nicotinic α-BGTX-insensitive and α-BGTX-sensitive receptors, and nonselective serotoninergic receptors (5-HT)) showed that the studied molecules do not bind signiﬁcantly to these receptors (Table 14.6). [33].

14.2.5

Biological Effects of TDZDs

Tau Phosphorylation Treatment of SH-SY5Y cell culture with different TDZDs at different concentrations (5, 15, and 50 μM) resulted in a decrease in endogenous Tau phosphorylation [34]. Detection was carried out after 24 hours treatment by a speciﬁc ELISA sandwich method using a phosphospeciﬁc antibody that detects an epitope on Tau residue Ser396, a site speciﬁcally phosphorylated by GSK-3 in cells. Cell survival was determined in parallel by measuring the LDH release. Moreover Tau phosphorylation levels were determined by using the phospho-speciﬁc antibody AD-2 in sagittal brain sections from TDZDtreated animals. Tet/GSK-3β double-transgenic mice [35] were treated orally with two different TDZDs once daily. After three weeks of treatment, a decrease in Tau phosphorylation was conﬁrmed by western blot analysis on hippocampal slices [34]. These results prove the potential of TDZDs to be developed as therapeutic agents for the treatment of AD and other tauopathies through the inhibition of GSK-3 and Tau phosphorylation. Neuronal Death The PI3K/PKB signaling pathway is recognized as playing a central role in the survival of diverse cell types. PKB phosphorylates GSK-3 in response to insulin and growth factors, which inhibits GSK-3 activity and leads to the modulation of multiple GSK-3 regulated cellular processes. Several TDZDs showed a dose-dependent protection in cultured human neuroblastoma cells from death induced by blocking the PI3K/PKB pathway [36].

10 100 2 10 2 3 4 6 4 6 5 2 3 2

GSK-3 (IC50)

— — — — — — — — — — — — — —

Abl-K

Selectivity of TDZDs for GSK-3b

23 — 16 35 — — — — — — — 21 — 15

CAM-K II — — 25 — 16 29 12 33 17 17 36 14 40 19

EGF-TyrK

Note: Data presented are shown as percentage of binding at 10 μM of TDZD.

5 7 8 10 11 12 13 14 16 17 18 19 20 21

Compound

TABLE 14.5

— 24 20 19 59 72 68 45 55 36 67 61 64 50

IRK — — — — — — — — — — — — 10 —

MAPK

— — — — — — 11 — — — 13 — — 19

MEK-IK

— 19 35 27 74 70 — — 58 — 55 28 82 —

p56lck

IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLDIONES

265

— 19 21 13 — — — — — — — — 12 —

5 7 8 10 11 12 13 14 16 17 18 19 20 21

— — — 22 — 31 29 42 13 13 14 — 15 —

D2 — 20 21 13 — — — — — 64 — — — 12

AMPA 12 — 10 — 23 26 20 31 24 10 26 19 32 —

NMDA

Note: Data presented are shown as percentage of binding at 10 μM of TDZD.

α2

Proﬁ le of binding assays to different receptors

Compound

TABLE 14.6

— — 21 16 — — — — — — — — — —

M — — — — — 23 — — 26 20 — 18 — —

α-BGTX-Insens. — — 19 — — 13 — 11 — — — 12 14 —

α-BGTX-Sens.

— 11 32 — 46 27 24 47 32 47 29 28 29 22

5-HT

266 TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLDIONES

14.2.6

267

Structure-Activity Relationships

TDZD The interesting enzymatic proﬁ le found in these compounds led us to focus on the preliminary structure-activity relationships. The size of the substituent attached to N2 of the thiadiazolidinone ring appeared to be crucial for GSK-3β inhibition, with methyl moiety being the best substitution obtained in our set of tests (see compound 1 versus 2–5, or TDZD-8 versus 9 and 10). This fact suggested a steric hindrance in the enzyme. Nevertheless, the nature of the substituent of N4 seemed also important for inhibition. When comparisons are possible, potency is always enhanced by the introduction of an aromatic moiety, such as the phenyl, methylphenyl, or naphthyl groups (compounds 8 and 11–20), suggesting favorable hydrophobic interactions with the protein. As regards the alkyl substituents in this N4 position, only the smallest ones, such as ethyl moiety, turned out to be a GSK3β inhibitor (compound 1 versus 6 or 7). Substitution of the carbonyl group attached to the C3 position of the heterocycle by a thiocarbonyl fragment led to derivatives that substantially retained the inhibitory potency (compounds 22, 24, and 25 versus 2, 10, and 9, respectively) but decreased compound solubility and stability. Then, when the C5 carbonyl group was replaced by the bioisosteric imino moiety, a dramatic decrease in inhibition was observed (compounds 26–29). This fact suggests that the C5-carbonyl group is critical to binding. Maleimides The maleimide scaffold can be considered as structurally related to the TDZD scaffold in which two heteroatoms, the sulphur atom and the nitrogen atom directly attached to it, are eliminated. However, the planarity of the ring is preserved with the double bond between both carbon atoms. In general, these compounds are GSK-3 inhibitors at a micromolar level (see Table 14.2). Inhibitory activity increased when the nitrogen atom of the maleimide ring was substituted by any aromatic or ester substituent (compound 30 and 31 versus 32 to 34). Further the length of the linker between the aryl group and the heterocyclic nitrogen does not inﬂuence the inhibitory activity (compounds 35 to 38). Considering the nature of the atom that bridges the two carbonyl groups, the inhibitory activity decreases in the sequence N > C > O (compounds 30, 39, and 40). Finally, it is clear that the presence of carbonyl groups and the double bond between the two carbons attached to the carbonyl groups are prerequisites for inhibitory activity (compounds 42–45). Rhodanines and Thiazolidines These compounds differ from TDZDs by the presence of only one nitrogen atom in the ring. The GSK-3 inhibition data are set out in Table 14.3. The N-benzyl-rhodanine derivative (compound 51) was found to have the best inhibitory activity, therefore conﬁ rming the importance of the role played by the aromatic substituent at this position. However, the activity diminishes when the link between the aromatic moiety

268

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

and the nitrogen atom is longer (compound 52). More remarkable is that the presence of a thiocarbonyl group is a prerequisite for activity in these compounds, as its replacement by a carbonyl group (compare compound 51 to compound 54) proved detrimental. Dithiazolidinediones, Triazolidinediones, and Hydantoines 1,2,4Dithiazolidine-3,5-diones derivatives bear a sulphur atom instead of the nitrogen atom at position 2 in TDZDs. In contrast, in a triazolidinedione ring a nitrogen atom replaces the sulphur atom present in TDZDs. Finally, some hydantoine compounds, where the sulphur atom is replaced by carbon, were also included in the study. The results of the biological assays against GSK-3 (Table 14.4) corroborated our previous ﬁ ndings. An aromatic substituent attached to the nitrogen atom, ﬂanked by the two carbonyl groups, appeared to be important for inhibitory activity (compound 57 versus 58 and 59). However, no such activity was present in the triazolidindione and hydantoine compounds, and this suggests a crucial role for the sulphur atom of the TDZD scaffold in modulating the inhibitory activity against GSK-3. 14.2.7

CoMFA and Mapping Studies

The CoMFA methodology [37], in conjunction with mapping studies, were used to gain insight into the molecular determinants that mediate the GSK-3 inhibitory activity of these compounds. The preceding data identiﬁed the important role played by the nature of the heteroaromatic ring as well as by certain substituents in mediating GSK-3 inhibition. To further explore the relationship between the chemical structure and the biological activity of TDZDs, a 3D-QSAR study was carried out by using CoMFA methodology. From this study and through the inspection of the relative contributions of steric and electrostatic molecular ﬁeld, it seems that the inhibitory activity in TDZD and related compounds is modulated by a subtle balance between the two ﬁelds. Because of the availability of X-ray crystallographic data for GSK-3, a mapping study was performed using the docking module of the CMIP program [38]. The CMIP analysis was performed on TDZD-10 and allowed us to identify two potential binding sites. One of them is located in the vicinity of the activation loop, near residues Arg96, Arg180, and Lys205 (the pocket on the right side of the enzyme as shown in Fig. 14.4). There appeared to be a good shape complementarity between the binding pocket and the ligand, which adopted an eclipse-like conformation with the two phenyl rings pointing toward the same face of the TDZD heteroaromatic ring. In this conformation, cation-π interactions formed between the phenyl rings and the positively charged Arg92 and Arg96 residues. The binding of TDZDs to this pocket, which may explain the non–ATP-competitive nature of these inhibitors, can make the

IDENTIFICATION OF 2,4-DISUBSTITUTED THIADIAZOLDIONES

269

Figure 14.4 Preferred binding sites determined from CMIP mapping studies for TDZD inhibitor 10 onto the X-ray crystallographic structure of GSK-3 (PDB entry 1I09). See color plates.

proper alignment of the substrate in the binding site difﬁcult and thereby lead to inhibition of the phosphorylation mechanism. An alternative binding site is found in the ATP binding cavity (the pocket on the left side of the enzyme as shown in Fig. 14.4), where the ligand adopts an extended conformation. This recognition mode seems not a priori to justify the non–ATP-competitive nature of TDZDs. However, considering the sensitivity of the GSK-3 inhibitory data to the nature of the heteroaromatic ring and, in particular, the need for the sulphur atom in the TDZD scaffold for inhibitory activity, one might reinterpret the electrostatic requirement found in the CoMFA analysis in terms of the chemical susceptibility of TDZDs to react with the thiol group of Cys199, which lies close to the TDZD heteroaromatic ring in some of the structures docked into the ATP-binding site. This ﬁ nding would explain the apparent GSK-3 speciﬁcity exhibited by TDZDs. 14.2.8

Second Generation of TDZDs

In order to check the inﬂuence of carbonyl groups on biological activity, different 1,2,4-thiadiazole derivatives were prepared in which the carbonyl

270

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

R3 N

O R1

N

N S

R2

5-imino-1,2,4-thiadiazol-3-one

R N

O R'

O

H N

N

Ph HBr Ph

N S

R1

3-alkyl-5-amino-1,2,4-thiadiazol

N S

TDZD R1

H N

N N S

H N

O R2

3,5-diamino-1,2,4-thiadiazol

Figure 14.5

R1

N N S

H N

R2

5-amino-1,2,4-thiadiazol-3-one

Second generation of TDZD.

groups were substituted by amino and alkyl groups (Fig. 14.5) [39]. The common ﬁ nal step employed for the synthesis of these last compounds was the oxidative cyclization using the corresponding intermediates in each case. Therefore for the families of 5-amino-1,2,4-thiadiazol-3-one and 5-imino1,2,4-thiadiazol-3-one there were the corresponding tiobiourets, and for the 5-amino-3-alkyl-1,2,4-thiadiazol the imidoylurea system and for the 3,5diamino-thiadiazol the corresponding ureas. After the biological evaluation against GSK-3 of all the previously synthesised derivatives, it is worth mentioning that 5-amino-1,2,4-thiadiazol-3-one derivatives together with 3-amino-5-alkyl-1,2,4-thiadiazol showed biological activity values against GSK-3 in the micromolar range. As regards the 5amino-1,2,4-thiadiazol-3-one family, a preliminary structure activity relationship, shows that the best prerequisites for the biological activity were found when small substituents were located in position 2 and hydrophobic substituents were located in position 5. With respect to the 3-amino-5-alkyl-1,2,4thiadiazol derivatives a more broad range in terms of structural requirements can be assumed for biological activity because the 5 amino position can be occupied by groups of a very different chemical range.

14.3 THERAPEUTIC POTENTIAL OF 2,4-DISUBSTITUTED THIADIAZOLIDINDIONES 14.3.1

Drug-like Properties

Having identiﬁed the family of TDZDs as non-ATP competitive GSK-3 inhibitors, the different drugability properties of some of the compounds above were explored to select potential candidates for development.

271

THERAPEUTIC POTENTIAL OF 2,4-DISUBSTITUTED TDZDs

In general, drug-like properties are considered to be those that demonstrate desirable pharmacokinetic and pharcodynamic (PK/PD) properties [40] independently of pharmacological target or indication [41]. In the case of the TDZDs family, the drug-like properties were conﬁ rmed, using not only theoretical methods but in vitro and in vivo models. The Lipinski rule of ﬁve is a good approach to estimating oral bioavailability or permeability [42]. The calculation of these basic chemical properties for some selected TDZDs indicated that they are within the limits of a promising drug. Additionally a neural network model [43] was used to predict both oral absorption and BBB permeability of TDZDs. These data were further veriﬁed experimentally using artiﬁcial membrane methodology [44–45] (Table 14.7). The potential candidates possessing the best properties were explored in vivo for absorption and blood-brain barrier permeability. The results showed that some compounds have sufﬁcient plasma levels and crossed the blood-brain barrier to be considered good candidates for drug development. In addition the results obtained using human microsomes, as well as both Cyp2D6 and Cyp3A4, showed that some of the chemicals were stable and had no inhibitory effects on the selected cytochromes (Table 14.8) [33]. Moreover a screening approach based on a reduced Ames assay (Table 14.9) and on an in vitro micronucleus test was performed using TDZD-10. The results were negative in these assays [46], pointing to a safe genotoxicity proﬁ le for TDZDs. Taken together, all the results showed that TDZDs possess a good drug proﬁ le for further development.

TABLE 14.7 Oral absorption and BBB permeability values approached by experimental in vitro model and in silico model In vitroa

In silicob

Compound

%Tx 10 4 (seg−1)

Predicc Oral Absorption

%Tx 10 4 (seg−1)

Predicc. BBB

Oral Absorption (%)

LogBB

5 10 19 21

21.85 30.87 26.32 —

OA+ OA+ OA+ —

22.80 10.30 23.43 23.04

CNS+++ CNS++ CNS+++ CNS+++

97.12 98.76 100 97.36

CNS+ CNS+ CNS+ CNS+

Oral Absorption Model: T% × 10 4 (seg−1) = 100(A R · V R /A D · V R); T% > 9.0 = OA > 80%. Blood-Brain Barrier Permeability Model: T% × 10 4 (seg−1) = 100(A R · V R /A D · V R); T% > 7.62 = CNS+. b Oral Absorption Model: Training set (Tsar) = 28; network architecture = 4 × 3 × 1; r 2 = 0.84; r 2cv = 0.63. Blood-Brain Barrier Permeability Model: Training set (Tsar) = 45; network architecture = 4 × 4 × 1; r 2 = 0.81; r2cv = 0.64; logBB > −0.3 = CNS+. a

272

TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

TABLE 14.8 Compound 5 10 19 21

ADME studies in vivo in mouse and in vitro in microsomes Plasma Levels Yes Yes Yes Yes

BBB Maxim Life Penetration (h) ND Yes ND Yes (i.v. only)

4 1 1 3

CYP2D6 (% Inhibition) 3 25 30 6

CYP3A4 (% Inhibition) 15 58 65 14

TABLE 14.9 Ames Screening Study: The TDZD-10 do not increase the number of revertants in S. Typhimurium Substance DMSO

Dilutions

Positive controls

Dose/Level μg/Plate

TA98

100 μl 0.32 1.6 8 40 200 1000 5000 Compound Dose level Mean ± SD

27 ± 6 26 ± 5 20 ± 2 19 ± 4 20 ± 6 12 ± 9 Toxic Toxic 2NF 5 μg 1067 ± 45

TDZD-10 TA100 120 ± 11 112 ± 9 110 ± 8 104 ± 4 119 ± 35 83 ± 21 Toxic Toxic NaN3 2 μg 749 ± 29

TA102 304 ± 13 308 ± 10 361 ± 28 310 ± 31 408 ± 85 390 ± 16 Toxic Toxic GLU 25 μg 562 ± 17

Note: A reduced screening version of the regulatory Ames assay with three S. Typhimurium strains (TA98, TA100, TA102) was used. The experiment was performed both in presence and absence of exogenenous metabolism (Aroclor 1254-induced S9-rat liver fraction). The plateincorporation method was used (OCDE Guideline nº471; 21 July 1997)

14.3.2

IGF-1 Mimetic Activity

IGF-1 inhibits burn-induced muscle proteolysis and dexamethasone-induced proteolysis by multiple mechanisms, including PI3K/Akt-mediated inactivation of GSK-3β and Foxo transcription factors. The GSK-3β inhibitors, LiCl and TDZD-8, reduced protein breakdown in both models (muscles and cultured L6 myotubes) in the same way as IGF-1 [47–48]. This evidence suggests that GSK-3β could be investigated in the future as a target for the inhibition of muscle wasting as well. 14.3.3

Apoptosis Modulation

Myeloma cell growth is regulated through the Akt/GSK-3/forkhead signaling pathway [49]. Disruption of this pathway by inhibiting downstream signaling

THERAPEUTIC POTENTIAL OF 2,4-DISUBSTITUTED TDZDs

273

protein GSK-3 is sufﬁcient to induce growth suppression and apoptosis. Both dose-response and time-course studies indicated that TDZD-8 is a potent inhibitor of myeloma cell growth and rapidly induces apoptosis at lower concentrations compared with LiCl and SB216763 [50]. Inhibition of GSK-3 activity by small-molecule compounds such as TDZD is effective in preventing inactivation of transcription factors that regulate proapoptotic and cell cycle inhibitor genes. Moreover these small-molecule compounds may provide a means of targeting downstream signaling elements in the IL-6 signaling cascade, which is likely to be more selective in their action. A pivotal signaling network in colorectal cancer cells involves GSK-3. Recent studies showed that acute ablation of GSK-3 in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. To test the suitability of this GSK-3-p53 axis for novel cancer therapeutic strategies, TDZD-8 was used reproducing the upregulation of p53 and the activation of p53-dependent apoptosis [51]. This strategy may provide a novel rational approach for the treatment of colorectal cancer patients retaining wild-type p53. 14.3.4

Anti-inﬂammatory Effect

GSK-3β plays a pivotal role in the regulation of the activation of NF-kB. The effect of TDZD-8 was investigated in vivo in a rat model of septic shock (with systemic inﬂammation and organ injury) caused by co-administration of lipopolysaccharide (LPS) and peptidoglycan (polymicrobial sepsis). The potent and selective inhibitor of GSK-3β, TDZD-8, 1 mg/kg administrated intraveneously 30 minutes before the co-administration of LPS, reduced the systematic inﬂammation associated with endotoxic shock in vivo [52]. The mechanism for the observed beneﬁcial effects of TDZD-8 may involve the inhibition of Ser536 residue on p65, prevention of the activation of NF-κB, and/or reduction of the expression of NF-κB-dependent proinﬂammatory genes. GSK-3β inhibition is proposed as a new strategy for the prevention or treatment of organ injury/dysfunction associated with sepsis, shock, and other conditions associated with local or systematic inﬂammation. In addition, administration of TDZD-8 (0.1, 0.33, or 1.0 mg/kg subcutaneously, b.i.d, for three days) caused a dose-dependent reduction of colonic inﬂammation induced by intracolonic trinitrobenzene sulphonic acid (TNBS) in rats assessed after three days, both in the area of macroscopic involvement and in a score using a 0 to 10 scale [53]. The increase in myeloperoxidase activity, an index of neurotrophil inﬁ ltration into the TNBS-induced inﬂamed colon, and the increase in the levels of the proinﬂammatory cytokine, TNF-α, were signiﬁcantly inhibited by TDZD-8. These ﬁ ndings demonstrate that the selective GSK-3β inhibitor TDZD-8 reduces inﬂammation and tissue injury in a rat model of acute colitis by an underlying mechanism of action probably involving the downregulation of NF-κB activity implicated in the generation of pro-inﬂammatory mediators.

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TDZD’S: SELECTIVE AND ATP NONCOMPETITIVE GSK-3 INHIBITORS

However, the relation between GSK-3 and the NF-κB pathway has not been fully characterized. In a recent study on human endothelial cells, it has been observed that the increase in NF-κB DNA binding activity induced by TNF-α was signiﬁcantly suppressed by GSK-3 inhibition [54]. In addition TDZD-8 completely blocked IkB-α degradation by TNF-α, without affecting phosphorylation of IKKβ. Because both IKKβ-dependent phosphorylation and subsequent ubiquination are necessary for NF-κB activation, it appears that GSK-3 regulates NF-κB activity through the ubiquitin-proteosome system, not through the IKKβ pathway. These results demonstrate an essential and novel signaling pathway leading to endothelial cell activation in response to pro-inﬂammatory cytokines. Hence GSK-3 blockade by lithium or newly developed speciﬁc inhibitors, including TDZD-8, could be used as a novel therapeutic strategy for atherothrombotic vascular diseases such as acute coronary syndromes and ischemic stroke through the reduction of thrombus formation, vascular inﬂammation, and ischemic organ damage.

14.3.5

Neuroprotectant Properties

Accumulated evidence points to GSK-3 as a critical intermediate in proapoptotic signaling cascades that are associated with neurodegenerative diseases, thus providing a potential target site amenable to pharmacological intervention (see Chapter 9 of this volume). Preliminary in vitro studies suggest potential neuroprotective effects of TDZDs against several insults, such as 6-OHDPA, lipopolysaccharide (LPS), stausporine-induced apoptosis, and glutamate. Pretreatment with TDZD-8 and lithium eliminate 6-OHDA neurotoxicity in a human dopaminergic neuronal cell line (SH-SY5Y) and in cultured rat cerebellar granule neurons (CGNs), indicating that GSK-3 is a key component of the proapoptotic signaling cascade [55]. TDZD-10 and TDZD-18 show neuroprotective activity in primary cortical neurons. These compounds protect cortical neurons from apoptosis induced by the cell-free medium from LPS-activated microglial cultures. In addition TDZDs protected cortical neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, suggesting that these compounds are potent neuroprotective agents against a wide variety of neuronal insults [56]. Neuroprotection by TDZDs was coincident with a decrease in cytokine, COX-2, and iNOS expression as well as NO production. Accumulating data indicate that in addition to glial cells, neurons can express iNOS [57], which can produce the neurotoxic NO [58]. The effects of TDZDs on neuroprotection and inhibition of glial activation by LPS stimulation could be mediated by activation of the PPARγ nuclear factor. In fact TDZD-10 and TDZD-18 can activate a reporter construct containing consensus PPARγ binding sites, and this activation is further enhanced by 9-cis-RA, behavior characteristic of a classical PPARγ / RXR permissive heterodimer [59]. Moreover the neuroprotective effects

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of these TDZDs are suppressed by GW9662, a speciﬁc antagonist of PPARγ. Collectively, these results indicate that TDZDs can be very effective neuroprotective and anti-inﬂammatory compounds in neuronal cells through, at least in part, activation of the nuclear receptor PPARγ. This study suggests possible therapeutic uses for TDZDs in certain brain disorders, such as multiple sclerosis, as well as Parkinson’s and Alzheimer’s diseases, where inﬂammatory responses play a major role. 14.4

CONCLUDING REMARKS

The 2,4-disubstituted thiadiazolidindione (TDZDs) are described as the ﬁ rst ATP-noncompetitive GSK-3 inhibitors. After an extensive SAR study of different heterocyclic families, TDZDs were revealed as the appropriate scaffold for the selective inhibition of GSK-3. The mappings permit us to hypothesize two binding modes, which in turn might imply relevant differences in the mechanism that underly the inhibitory activity of TDZDs. Further studies, including directed mutagenesis, proteomic analysis, and determination of crystal complexes structures, should help clarify the mechanistic implications provided by our results. TDZDs are small synthetic heterocycles with favorable ADME-Tox drugable properties. They have been proposed as new drugs for the potential treatment of unmet diseases mediated by GSK-3 such as Alzheimer’s disease and other neurodegenerative disorders where Tau phosphorylation plays a key role, inﬂammatory acute and/or chronic diseases, and cancer. TDZDs’ therapeutic potential has been proved using a rat model of septic shock and acute colitis in which inﬂammation and tissue injury was reduced as a result, and also using a GSK-3β conditional transgenic mice (Tet/GSK-3β) in which after prolonged treatment a signiﬁcant decrease was observed in Tau phosphorylation within both the hippocampus and cortex areas where the transgene is expressed. Clearly, the clinical utility of these molecules awaits human trials. REFERENCES 1. Castro A., Martínez A. (2000). Inhibition of tau phosphorylation: A new therapeutic strategy for the treatment of Alzheimer’s disease and other neurodegenerative disorders. Exp. Opin. Ther. Patents, 10, 1519–1527. 2. Martínez A., Castro A., Dorronsoro I., Alonso M. (2002). Glycogen synthase kinase 3 inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inﬂammation. Med. Res. Rev., 22, 373–384. 3. Dorronsoro I., Castro A., Martínez A. (2002). Inhibitors of glycogen synthase kinase-3: Future therapy for unmet medical needs? Exp. Opin. Ther. Patents, 12, 1527–1536.

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15 3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GLYCOGEN KINASE SYNTHASE 3 (GSK-3) INHIBITORS Jason Witherington Department of Medicinal Chemistry, Neurology & Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline RhD, New Frontiers Science Park, Harlow, Essex, UK

Contents 15.1 15.2 15.3 15.4

Identiﬁcation of 5-Aryl Pyrazolopyridines Identiﬁcation of 5-Aryl Pyrazolopyridazines Structure-Based Design of 6-[Het]Aryl Pyrazolopyridines Cytochrome P450 and In vitro Microsomal Clearance Data for 5-Aryl Pyrazolopyrid[az]ines and 6-[Het]Aryl Pyrazolopyridines 15.5 In vivo Efﬁcacy Data for a Selective, Orally Bioavailable GSK-3 Inhibitor 15.6 Emerging Templates Containing a 3-Amino Pyrazole Scaffold

15.1

IDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDINES

Although many classes of kinase inhibitors have been identiﬁed from highthroughput screening (HTS) campaigns, a complementary approach toward lead identiﬁcation has been based around focused screening sets and employing the use of homology models in order to carry out de novo design. Recently the X-ray structure of GSK-3β has been reported [1], enabling the possibility for structure-based drug design. Based on the extensive X-ray ligand bound Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

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3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

protein kinase structures available, a widely accepted pharmacophore model was proposed by Furet in 1995 [2]. Since this time the model has been employed in the de novo design of many inhibitor series [3]. The model divides the ATP binding pocket into a number of smaller regions (Fig. 15.1). The adenine region is mostly hydrophobic in character, and it is in this region that ATP forms two important hydrogen bonds with the hinge region. In general, kinase inhibitors form at least one hydrogen bond with the hinge region. The sugar pocket, in contrast, is mainly hydrophilic in nature, and it has successfully been employed to gain potency and selectivity. While not utilised by ATP, hydrophobic region 1 varies in shape and size due to the presence of a “gatekeeper” residue. This pocket has been extensively employed

H

Hinge region

N

O N

H N

hydrophobic region 1

O

H N

H

H

N

O

N

N

-

O

N O

hydrophobic region 2

O

P O

HO

-

O

O

O

adenine region "sugar" pocket

6

N

4 N 7

O P

O O

OH

5

O

P

-

-

phosphate binding region

NH2 3 N H 1

N2

1

Figure 15.1

Protein kinase pharmacophore model.

IDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDINES

283

by medicinal chemists to impart both potency and selectivity [2]. In contrast, hydrophobic region 2, which is not utilized by ATP, is more of a hydrophobic slot. This pocket has also been utilized to great effect in order to gain potency and impart selectivity. The ﬁ nal region is the phosphate binding region that has high solvent exposure. Although binding in this region does not normally lead to signiﬁcant increases in potency, it can be used to impart selectivity due to the large number of nonconserved amino acids [2]. A pharmacophore search of the SmithKline Beecham compound database based on the ability of the prospective ligands to form at least one possible hydrogen bonding interaction with the hinge region and bind to at least one of the hydrophobic pockets of the ATP binding pocket, led to the identiﬁcation of pyridazine 1 as a potent inhibitor of human GSK-3α (IC50 250 nM) [4]. In order to understand the binding interaction with the active site, pyridazine 1 was docked into a GSK-3α homology model (constructed from the crystal structure of the highly homologous cyclin dependant kinase-2 (CDK2) (1hcl)) using the program GOLD. The highest scoring binding mode suggested a three-point hydrogen bonding interaction with the hinge region of the ATP binding site (Fig. 15.2). Interestingly, although there is the possibility of an alternative three-point binding mode, together with two possible twopoint hydrogen bonding binding modes, the program did not score any of these alternative binding modes highly. Further analysis of the binding mode indicated that the phenyl ring at C-4 was orientated toward the front of the ATP binding site, suggesting that this group could be removed to simplify the lead structure. The phenyl ring at C-5 appeared to be orientated toward the large hydrophobic pocket at the rear of the active site. Although this hydrophobic pocket is a common feature to all kinases, due to subtle steric and electronic differences between kinases, it has been routinely exploited in order to gain potency and selectivity. Thus it was hypothesized that optimization at C-5 may increase GSK-3’s potency. The

O N

Asp133 H O

N

H

O

Val135

Tyr H N N H N

Pro136

O

H N

Lys85 N

N

H N+ H H -

H

O

O

Glu97

iLeu62

Figure 15.2 GOLD docking of pyridazine 1 into the GSK-3-α homology model. See color plates.

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3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

amino group at C-3 appeared to be orientated toward a number of hydrophobic residues that exist at the solvent front of the ATP binding site, indicating this moiety could be readily functionalized in order to modulate potency or the physiochemical properties of the inhibitor if required. The nitrogen atoms in the six-membered ring at positions 6 and 7 did not appear to be critical for binding. Thus further modiﬁcation of the parent scaffold appeared possible, without compromising potency. To gain conﬁdence in the results from the automated ligand docking exercise, a small set of targeted analogues based around 1 were designed and prepared (Table 15.1). As predicted, removing the aromatic group at C-4 (cf. 1, 2) and the nitrogen at position 6 (cf. 2, 3) could be tolerated with only minor loss of potency. Removal of the nitrogen at position 7 appeared to be less well tolerated (cf. 2, 5), however, since it was noted that removal of the phenyl ring at the C-4 leads to poor inhibitor solubility, the absence of a group at C-4 could also account for this modest reduction in activity. Finally, from the limited data available, the aromatic group at C-5 appeared critical for potency (cf. 3, 4), consistent with it binding in the hydrophobic pocket at the back of the ATP binding site. Having simpliﬁed the lead structure, by removing the nitrogen at position 6 and the phenyl group at C-4, a number of compounds were synthesized to further validate the three-point H bonding pharmacophore (Table 15.2). Removal of the H bond donor at N-1, via methylation of the pyrazolo ring or substitution of nitrogen for oxygen (cf. 3 with 6 and 7), lead to a dramatic loss of potency. Unfortunately, this modiﬁcation does not discriminate between either the two- or three-point binding modes. Evidence to support the threepoint binding modes came from the ﬁ nding that removal of the postulated H bond acceptor at N-2 lead to poor GSK-3 activity (8, IC50 > 5000 nM) [8]. Interestingly acylation of the nitrogen at N-3 afforded a modest increase in potency (9, IC50 > 291 nM) [9] and from the predicted binding mode it TABLE 15.1 analogues

Inhibition of hGSK-3-a by selected pyrazolopyrid[az]ine 2

R

NH2

1

R

X

N N H

Y

Compound

R1

R2

X

Y

GSK-3α, IC50 nM

1 2 3 4 5

Ph Ph Ph H Ph

Ph H H Ph H

N N CH CH N

N N N N CH

250 530 430 >5000 1260

285

IDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDINES

TABLE 15.2

Inhibition of hGSK-3-a by selected heterocycles

NHR Ph Y N Compound 3 6 7 8 9

X

X

Y

R

GSK-3α, IC50 nM

NH NMe O NH NH

N N N CH N

H H H H COMe

430 >5000 >5000 >5000 291 ± 73

TABLE 15.3 Inhibition of hGSK-3-a for representative C-3 analogues

NHR Ph N N

N H

Compound

R

GSK-3α, IC50 nM

10 11 12 13 14 15

COEt COnPr COiPr COcPent CONHEt SO2Me

43 ± 5 56 ± 6 19 ± 3 5±1 2810 3572

appeared that a further increase in potency could be obtainable through a lipophilic interaction with ileu62. In order to explore this possibility, amides at C-3 incorporating more lipophilic substituents (10–13) were prepared (Table 15.3). Increasing the size of the lipophilic substituent did indeed lead to dramatic improvements in potency, the cyclopentyl analogue (13) displayed a nearly 60-fold improvement over the corresponding methyl analogue (9). Replacing the amide moiety with a urea (14) reduced potency considerably, presumably due to formation of a six-membered intramolecular hydrogen bond between the N-2 atom of the pyrazole nucleus and the hydrogen of the urea, which would disfavor the proposed bioactive conformation. Replacing the amide moiety with a sulfonamide (15) moiety also led to a considerable loss in potency, presumably due to the conformational differences between amides and sulfonamides.

286

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

In order to probe the SAR around the C-5 position compound 17, in which the C-5 phenyl ring has been removed, was initially prepared and was subsequently shown to be a weak inhibitor of GSK-3 (Table 15.4). Since the phenyl ring at the C-5 position was shown to be critical for binding, the SAR around this position was also explored in greater detail. Interestingly the boronate 16, which was prepared to enable the rapid expansion of the C-5 SAR, displayed submicromolar potency, indicating that an aromatic group is not essential for activity. A range of hydrophobic groups were introduced at the C-5 position as were groups designed to explore H bonding interactions. The ﬂuorinated phenyl derivatives 18, 19, and 20 showed an increase in potency relative to 11 as did the 3-pyridyl analogue 22. The potency gained through the ﬂuorinated analogues could arise via either optimization of lipophilic contact with the back of the ATP binding pocket, or from an H bonding interaction with Lys85. From the predicted binding mode, the increase in potency observed for the 3-pyridyl analogue could be due to interaction with the conserved salt bridge (Lys85/Glu97). Analogues containing larger groups at the C-5 position, such as 24 and 25, were signiﬁcantly less potent, suggesting that excess steric bulk around this position is detrimental, again supporting the postulated three point binding mode whereby the C-5 substituent is in close proximity to the conserved salt bridge. Having identiﬁed a potent series of GSK-3α inhibitors, some of the compounds above were proﬁ led against a panel of more than 20 diverse kinases, including GSK-3β in order to gain an understanding of the selectivity issues of this series (Table 15.5) [5]. In general, excellent selectivity is obtained against the majority of the kinases on the panel, however signiﬁcant inhibition of cyclin dependant kinase 2 (CDK-2) was observed. This level of CDK-2 inhibition was not unexpected given the high degree of homology between the two kinases, especially around the ATP binding pocket. TABLE 15.4

Inhibition of hGSK-3-a for representative C-5 analogues

H N

O

R N N Compound 11 16 17 18 19 20

R

N H

GSK-3α, IC50 nM

Ph 56 ± 6 B(OCMe2CMe2O) 356 ± 129 H 2343 ± 135 2-F-Ph 18 ± 2 3-F-Ph 20 ± 3 2,3-DiF-Ph 7±1

Compound

R

GSK-3α, IC50 nM

21 22 23 24 25 26

2-Cl-Ph 3-Pyridyl 4-Pyridyl 4-Ph-Ph 2-Naphthyl 1-Naphthyl

27 ± 6 11 ± 2 443 ± 50 851 ± 51 169 ± 14 241 ± 8

Inhibition of hGSK-3-a by selected

H N

O

Z X Compound 11 27 28 29 30 31

15.2

N N H

Y

GSK-3β

7 5 11 19 81 90 4 2 0 5 95 99 0 10 13 13 99 94

Note: Values are %I @10 μM using 100 μM ATP (see [5] for kinases used and assay details).

TABLE 15.6 analogues

CDK2/Cyclin A

SAPK4

SAPK3

SAPK2b

SAPK2a

ROKa

PRAK

0 13 12 22 18 1 0 5 0 5 14 0 3 2 11 13 4 0

PKCA

0 0 5

PKBa

PKA

PDK1

4 13 0 0 1 0

PHOS.K

0 6 10 9 4 5 7 3 9 3 9 8

P70S6K

MSK1

MEK1

MAPKAP-K2

JNK

CKII

Chk1

4 0 0

AMPK

11 14 11 10 11 34 18 14 1 0 0 36 22 4 0 0 7 10

RSK-2

Compound

MAPK

Selectivity of pyrazolo[3,4-b]pyridines for GSK-3b

LCK

TABLE 15.5

SGK

287

IDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDINES

X

Y

Z

GSK-3α, IC50 nM

CH CH N N N N

N CH N CH CH N

CH CH CH CH N CPh

56 ± 6 99 ± 15 4±1 7±1 2697 691 ± 15

IDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDAZINES

With the initial SAR supporting the proposed binding mode, the SAR around the pyridine nucleus was reinvestigated in order to identify compounds with varying physiochemical properties and potentially improved selectivity proﬁ les against CDK-2 (Table 15.6) [6]. As predicted from the postulated binding mode, removal of the nitrogen at position 7 (cf. 11, 27) was well tolerated. Surprisingly, reintroduction of the nitrogen at N-6 (cf. 11, 28, 29) afforded a dramatic improvement in potency. This is in contrast to the early SAR, which suggested that the nitrogen at position 6 was not required for inhibitory potency. Since this ﬁ nding could not be explained from any of the putative binding modes, the amine

288

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

moiety of the original lead 1 was acylated in order to ascertain whether the SAR was consistent between series. Unfortunately, this modiﬁcation led to a reduction in potency (cf. 11, 31), although this reduction could be explained via a steric clash between the required bioactive conformation of the amide bond and the substituent at C-4. Further evidence supporting this rationale came from the ﬁ nding that the pyrimidine 30 is also a weak inhibitor of GSK-3, possibly due to a repulsion of the lone pairs between the nitrogen at position 4 and the carbonyl of the amide bond. In order to try and understand why introduction of nitrogen into the 6 position lead to an increase in potency, further analysis of the putative binding modes was undertaken. However, all attempts to rationalise this ﬁ nding based on the proposed binding modes proved unsuccessful. A rationale was ﬁ nally gained from the analysis of the X-ray structure of a series of 2-aminothiazoles co-crystallized with CDK-2 (Fig. 15.3) [7]. Interestingly in this case the 2-aminothiazole nucleus forms a similar three-point hydrogen bond with the hinge region of the ATP binding site, but perhaps more important, one of the ketone lone pairs is involved in a hydrogen bond to a structural water molecule [8]. An overlay of the 2aminothiazole with the proposed binding mode of a pyrazolopy-ridazine in the GSK-3 homology model suggested that the increase in potency may be due to the presence of a structural water molecule in GSK-3, which was absent in the homology model. Indeed, 2-aminothiazoles have recently been disclosed as potent GSK-3 inhibitors [9]. Thankfully, the three-point hydrogen bond binding hypothesis and the presence of structural water molecules was subsequently conﬁ rmed by the determination of a co-crystal structure of a pyrazolopyridazine bound to GSK-3 (Fig. 15.4). Importantly, from the X-ray structure all three hydrogen bonding interactions are less than 2.1 Å, indicating a strong hydrogen bonding network. The crystal structure contains an interesting lattice of water, whereby the nitrogen at the 6-position hydrogen bonds to a water molecule that is also involved in a hydrogen bonding interaction with an NH of a backbone aspartic acid residue and a

A

B

O Leu N Leu O

Glu

Phe

Glu H H

N

H N

N

H

S

O

H

C Glu O

H O

Val

N

Val O

H

O H N N

H N

N

N

Val O

H HN H N N N ON H N N H N S O O H N

Val

H

Figure 15.3 (A) X-ray of 2-aminothiazoles in CDK-2; (B) model of pyrazolopyridazines in GSK-3; (C) overlay of A and B.

H O

289

INDENTIFICATION OF 5-ARYL PYRAZOLOPYRIDAZINES

O Asp133 H O Val135 Pro136

N O

N

H

Glu97 N

O

H

H N

O

H2O

O H Tyr N N

N

-

H2O

N

H

Asp200

N

O

O

0 14 0 8 0 5

21 5 0 5 0 7 14 12 11 0

GSK-3β

CDK2/Cyclin A

SGK

SAPK4

SAPK3

SAPK2b

SAPK2a

ROKa

PRAK

PKCA

0 0 8 30

PKBa

0 2

PKA

PHOS.K

PDK1

5 0 9 16

P70S6K

0 0

MSK1

MEK1

RSK-2

MAPK

6 0 15 0 4 35 5 11

MAPKAP-K2

28 12 0 18 32 18 25 16

LCK

JNK

CKII

Chk1

Selectivity of pyridazine 28 and 32 for GSK-3b inhibition

AMPK

TABLE 15.7

Compound

Figure 15.4 X-ray co-crystallisation of a pyrazolo[3,4-b]pyridazine with GSK-3. See color plates.

0 0 90 99 0 12 45 93

second water molecule. The second water molecule is itself involved in a hydrogen bonding interaction with a conserved glutamic acid. Having identiﬁed an extremely potent series of GSK-3 inhibitors, attention was focused on improving the physicochemical characteristics of the pyrazolopyridazine series. In general, kinase inhibitor scaffolds have poor aqueous solubilities due in part to their ﬂat hydrophobic characteristic, which is required for efﬁcient binding in the purine binding pocket of the active site. In order to improve the water solubility of the pyrazolopyridazine series, the amide moiety was targeted as this group provided a suitable vector to access the solvent front. Previously, in the related pyrazolopyridine series, it had demonstrated that a butyramide group at position 3 was of optimal length for GSK-3 potency hence attachment of basic amine groups onto this side chain was investigated. Importantly, attachment of a basic amine group on to the butyramide side chain was tolerated, albeit with a ca 5 fold loss in potency (cf. 28, 32) (Table 15.8). A number of heterocyclic amines were also prepared (33, 34, 35) in order to probe for hydrogen bonding interactions. However, only modest improvements in potency were observed consistent with the binding mode. Increas-

290

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

ing the chain length also had no beneﬁts in terms of potency (cf. 34, 37). The modest improvement in potency observed when cyclizing the butyramide side chain to afford the piperidine analogue 36 was disappointing, given the increase in potency that had been observed for cyclic aliphatic side chains in the structurally similar pyrazolopyridine series. Although incorporation of a basic side chain into the molecule led to a small reduction in potency, in the related pyrazolopyridine series the potency of the inhibitors could be enhanced through incorporation of a 2,3-diF-Ph moiety at C-5, and hence this modiﬁcation was also explored in the pyrazolopyridazine series. Thankfully, incorporation of this group into the pyridazine series afforded a similar increase in GSK-3 potency to that observed previously (cf. 32, 38). The selectivity proﬁ ling of 33 was also determined (Table 15.7). A surprising ﬁ nding was identiﬁed whereby incorporation of a basic side chain at C-3 led to an excellent improvement in selectivity against CDK-2. In order to ascertain the degree of CDK-2 selectivity, K i’s were obtained for a small subset of inhibitors. From the data obtained it was clearly evident that incorporation of a diverse range basic side chains routinely resulted in an excellent improvement in selectivity (Table 15.9). In order to understand the origin of this selectivity, an overlay of the GSK-3 and CDK-2 crystal structures was performed. From this analysis it appeared that the origin of this selectivity was likely to be due to a steric/electronic clash between the basic side chain and a salt bridge, that is present in CDK-2 but not in GSK-3 (cf. 28, 32) (Fig. 15.5).

TABLE 15.8 side chains

Inhibition of hGSK-3-a by pyrazolopyridazines containing basic

R

1

N Compound 28 32 33 34 35 36 37 38

O

H N

R

2

N N

N H

R1

R2

GSK-3α, IC50 nM

Ph Ph Ph Ph Ph Ph Ph 2,3-DiF-Ph

Bu (CH 2) 3NMe2 (CH 2) 3pyrrolidine (CH 2) 3piperazinyl-N-Et (CH 2) 3morpholinyl 4-Piperidine-N-Me (CH 2) 4piperazinyl-N-Et (CH 2) 3NMe2

4±1 22 ± 2 11 ± 1 7±2 5±1 9±1 5±1 5±1

STRUCTURE-BASED DESIGN OF 6-[HET]ARYL PYRAZOLOPYRIDINES

TABLE 15.9

CDK-2 selectivity determination

R

H N

1

N Compound 39 40 32 41 42

291

R1 3-Pyridyl 2,3-DiF-Ph Ph 2,3-DiF-Ph 2,3-DiF-Ph

O R

2

N N

N H

R2 cyPr cyPent (CH 2) 3NMe2 4-Piperidine-N-Et CH 2Piperidine-N-Et

GSK-3α, Ki (nM)

CDK-2, Ki (nM)

CDK-2/ GSK-3

0.08 0.11 2.5 0.95 0.19

5 5 1695 450 540

62 45 678 474 2842

Figure 15.5 Overlay of the crystal structures of GSK-3 (green) and CDK-2 (white) with compound 32. See color plates.

15.3 STRUCTURE-BASED DESIGN OF 6-[HET]ARYL PYRAZOLOPYRIDINES In order to fully exploit the pyrazolopyrid[az]ine template futher, the ligand bound crystal structure was further analyzed to see whether modiﬁcation to the central template was possible. From this analysis it was evident that it might be possible to design inhibitors wherein the structural waters present in the binding site were displaced via insertion of a suitably functionalized

292

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

group, such as a para or meta phenol moiety at the 6-position of the pyrazolo[3,4-b]pyridine nucleus (Fig. 15.6) [10]. Indeed, GOLD docking studies conducted using the GSK-3 apo crystal structure and a range of C-6 containing phenol analogues appeared favorable. In order to test this hypothesis, a small set of functionalized C-6 aryl pyrazolopyridines was designed and synthesized (Table 15.10). As predicted from docking studies, the para phenol derivative 44, demonstrated a dramatic improvement in potency compared to the parent phenyl analogue 43. From modelling studies it was predicted that the phenol moiety was involved in both hydrogen bond donor and acceptor interactions. To test this hypothesis, the

O Asp133 H O 1.5Å 2.6Å

Val135

N

Pro136

O

N

H

Glu97

N

H

O

O H Tyr N N H N

-

O

OH N

O

H

N

Asp200 O

Figure 15.6 Gold docking run of a C-6 phenol pyrazolopyridines. See color plates. TABLE 15.10 Inhibition of hGSK-3-a by selected 6-aryl-pyrazolopyridine analogues

H N

O

N N

R Compound 43 44 45 46 47 48 49 50

N H

R

GSK-3α, IC50 nM

H 4-OH 4-OMe 3,4-Di-OH 3-OH 3-OMe 2-OH 2-OMe

425 8 >5000 8 12 125 76 1593

STRUCTURE-BASED DESIGN OF 6-[HET]ARYL PYRAZOLOPYRIDINES

293

para methoxy analogue 45 was prepared and was shown to be inactive, although this could be a result of a steric clash with Glu97 rather than the loss of the hydrogen bond donor interaction. Interestingly the meta phenol analogue 47 displayed comparable potency to the para phenol analogue 44, supporting the modeling analysis whereby the meta phenol moiety was predicted to be involved in the same hydrogen bonding network as the para phenol isomer. Support for this rationale comes from the ﬁ nding that analogue 46, which has a hydroxyl moiety at both the 3 and 4 positions, displays comparable potency to both 47 and 44. Further support for the H bond donor/ acceptor rationale comes from the ﬁ nding that the 3-methoxy analogue 48, is 10-fold less potent than the parent phenol analogue 47, suggesting an additional hydrogen bonding interaction is made with the hydrogen of the phenol moiety. Although the ortho phenol analogue 49 is more potent than the parent phenyl analogue 43, it is signiﬁcantly less potent than either the para or meta analogues. Having investigated the SAR around the C-6 position, the C-5 position was also explored since in the 5-aryl pyrazolopyridine series it had been demonstrated that a group at this position was essential for inhibitory potency (Table 15.11). Interestingly insertion of a phenyl group at the C-5 position had no beneﬁcial effect on inhibitory potency (cf. 44 and 51, 55 and 56). The C-5 SAR differences observed between the 5-aryl pyrazolopyridazine series and the 6-aryl pyrazolopyridine series may be as a result of a change in the torsion angle of the C-5 aryl group due to the presence of the C-6 substituent. Support for this hypothesis comes from analysis of the ligand-bound crystal structures of the 5-aryl pyrazolopyridazine series where the C-5 substituent is co-planar to the pyrazolopyridazine nucleus. Interestingly, however, insertion of bromine at the C-5 position afforded a four to six fold improvement in potency Table 15.11 Inhibition of hGSK-3-a by selected C-5 substituted 6-arylpyrazolopyridine analogues O R

H N

2

N N

1

R Compound 44 51 52 53 54

R1

R2

4-OH 4-OH 4-OH 4-OH 4-OH

H Ph Br Cl Me

GSK-3α, IC50 nM 8 24 <2 <2 6

N H

Compound

R1

R2

GSK-3α, IC50 nM

55 56 57 58 59

H H H H H

H Ph Br Cl CN

425 415 67 232 87

294

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

(cf. 44 and 52, 55 and 57). Several hypotheses could account for the increase in potency observed. First, the group at the C-5 position also occupies the ATP binding site, and thus the inhibitor has greater surface contact with the enzyme. Second, there is a speciﬁc hydrophobic interaction with the bromine atom at the C-5 position and the ATP binding site. Third, the substituent at the C-5 position modulates the acidity of the pyrazolo ring, and thus also optimizes the hydrogen bonding interaction with Asp133 of the hinge region of the ATP binding site. Support for the increase in potency observed being due to the modulation of acidity comes from the ﬁ nding that the nitrile analogue 59 displays comparable potency to the bromo analogue 57. Previously, in the structurally related 5-aryl-pyrazolopyridazine series, incorporation of a basic side chain into the C-3 position afforded improvements in solubility, selectivity and metabolic clearance. In order to develop the SAR some more, this modiﬁcation was explored in the 6-aryl-pyrazolopyridine series. Attachment of basic amines onto the side chain of the amide moiety was tolerated in most cases without signiﬁcant loss of potency (Table 15.12). This SAR is consistent with previous ﬁ ndings, suggesting a similar binding mode to the 5-aryl pyrazolopyridazine series where crystallographic studies show that the amide group is orientated out of the active site toward the solvent accessible front. Because of the high degree of homology between GSK-3 and cyclindependant kinase 2 (CDK-2) and the ﬁ nding that the structurally related 5aryl pyrazolopyid[az]ines can display excellent CDK-2 inhibitory potency, a range of a 6-aryl-pyrazolo[3,4-b]pyridines were screened against CDK-2 (Table 15.13). Incorporation of a hydroxyl group at either the para or meta

TABLE 15.12 Inhibition of hGSK-3-a by 6-aryl-pyrazolopyridines containing basic side chains

R

H N

2

O R

3

N R Compound 60 61 62 63 64 65

N

1

N H

R1

R2

R3

GSK-3α, IC50 nM

H H 4-OH 4-OH 4-OH 3-OH

Br Br Br H Br H

4-Piperidine-N-Me (±)-Pyrrolidine-N-Bn (CH 2) 3piperazinyl-N-Et 4-Piperidine-N-Me 4-Piperidine-N-Me (CH 2) 3piperazinyl-N-Et

383 15 4 42 <2 21

STRUCTURE-BASED DESIGN OF 6-[HET]ARYL PYRAZOLOPYRIDINES

TABLE 15.13 analogues

295

Inhibition of hGSK-3a and CDK-2 by selected pyrazolopyridine

R

H N

2

O R

3

N R Compound 44 47 51 52 57 66 67 68

1

R1

R2

4-OH 3-OH 4-OH 4-OH H 3-Br-4-OH 3-Cl-4-OH 4-OH

H H Ph Br Br H H Br

N

N H R2

cyPr cyPr cyPr cyPr cyPr cyPr cyPr (CH 2) 3piperazinyl-N-Et

GSK-3α, IC50 nM

CDK-2, IC50 nM

5 8 16 <2 67 5 7 3

8 60 5 5 >10,000 2 2 60

position of the phenyl ring at the C-6 position, afforded not only excellent GSK-3 potency (cf. 44, 47, and 57) but also a concomitant increase in CDK-2 inhibition. Attempts to introduce CDK-2 selectivity by variation of the group at C-5 proved unsuccessful (cf. 44, 51, and 52). Given that incorporation of a basic side chain into the 5-aryl-pyrazolopyridine series imparted excellent improvements in CDK-2 selectivity, this modiﬁcation was also explored in the 6-aryl-pyrazolo[3,4-b]pyridines series. Incorporation of this modiﬁcation did indeed afford inhibitors with somewhat improved selectivity (e.g. 68); however, the absolute level of CDK-2 inhibitory potency still remained high. Next, efforts to introduce selectivity by introduction of functionality next to the hydroxyl moiety also proved unsuccessful (cf. 44, 66, and 67). From modelling studies the increase in both GSK-3 and CDK-2 potency, by introduction of the hydroxyl group, can be rationalized from the ﬁ nding that Glu97 and Asp200, the proposed residues interacting with the hydroxyl moieties, are conserved in all kinases. Further support for this binding hypothesis comes from proﬁ ling the phenols 52 and 44 against a panel of kinases where a more promiscuous inhibitory proﬁ le, compared to the parent analogue 57, is observed (Table 15.14). Although selectivity against CDK-2 was a generic issue for the phenol analogues, the phenyl analogue 57 possessed an excellent overall selectivity proﬁ le. Therefore a range of alternative C-6 substituents were explored in order to identify a potency enhancing group that was also selective for GSK-3. For synthetic expediency, SAR around the C-6 position work was carried out

296

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

GSK-3β

CDK2/Cyclin A

DYRK1A

SAPK4

SAPK3

SAPK2b

SAPK2a

ROKa

PRAK

PKCA

PKBa

PKA

PHOS.K

PDK1

P70S6K

MSK1

MEK1

MAPKAP-K2

MAPK

LCK

Selectivity proﬁ ling of pyrazolopyridines 57 and 52 and 44

JNK

CKII

Chk1

AMPK

Compound

TABLE 15.14

57 0 28 31 0 49 0 12 10 5 0 20 3 3 0 0 21 8 79 11 1 0 28 25 87 52 57 66 3 15 89 45 19 5 58 14 59 30 19 17 62 1 4 41 59 62 61 80 100 100 44 56 0 3 12 70 29 14 12 65 8 31 80 20 17 83 41 15 10 58 69 92 91 90 Note: Values are %I @10 μM using 100 μM ATP (see [5] for kinases used and assay details).

on the indazole nucleus, since it had previously been shown to possesses similar potency to pyrazolo[3,4-b]pyridine (Table 15.15) [11]. To validate the strategy, the phenyl and phenol analogues 69 and 70 were prepared. As predicted, an increase in potency against GSK-3 and CDK-2 was observed for 70 when compared with 69. Although the indolyl analogue 71 displayed excellent GSK-3 potency and CDK-2 selectivity, in general, attempts to identify alternative phenol isosteres (e.g., 72–77) proved unsuccessful. Attempts to optimize the lipophilic interaction of the phenyl ring (e.g., 78–83) also proved generally unsuccessful. Interestingly introduction of small heterocyclic substitutents at the C-6 position consistently afforded analogues (e.g., 84–88) with greater GSK-3 potency than the corresponding phenyl analogue 69, and in some cases the SAR suggested that selectivity against CDK-2 might be achievable (e.g., 85, 86). Since such heteroaromatic groups at C-6 were postulated not to interact directly with Glu97 or Asp200, the promising C-6 SAR in the indazole series was transferred into the more potent 5-bromo pyrazolopyridine series (Table 15.16). As predicted, incorporation of bromine into the 5-position of the 6heteroaryl pyrazolopyridine nucleus afforded very potent inhibitors of GSK-3 that were selective against CDK-2 (e.g., 90, 92, 94). However, the magnitude of the potency enhancement is particularly noteworthy given this modiﬁcation affords a nearly 25-fold increase in potency without compromising CDK2 selectivity (cf. 91 and 92). Exploring the SAR at the C-3 position also conﬁ rmed previous ﬁ ndings from related series in that lipophilic amides are preferred with the observation that substituting a cyclopropyl group for a cyclopentyl can afford a 5- to 10-fold improvement in potency (cf. 89, 90, 93, 94), and importantly, incorporation of water-solublizing groups was also tolerated at the 3-amino position, affording potent GSK-3 inhibitors while retaining excellent CDK-2 selectivity (e.g. 95, 96). The SAR around the C-5 and C-3 substituents was consistent with the binding hypothesis derived from the automated ligand docking of the phenol derivatives.

69 70 71 72 73 74 75 76 77 78

Compound

Ph 4-OH 5-Indolyl 6-Quinolyl Ph-4-SO2NH 2 Ph-3-SO2NH 2 Ph-3-NHSO2Me Ph-4-NHSO2Me 5–1H-pyrid-2-one 2-F-Ph

R 529 15 42 >1000 >1000 481 >1000 >1000 >1000 >1000

GSK-3α, IC50 nM

R Compound 79 80 81 82 83 84 85 86 87 88

>1000 5 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000

N

O

CDK-2, IC50 nM

N H

H N

TABLE 15.15 Inhibition of hGSK-3a and CDK-2 by selected indazole analogues

2, 5-Dif-Ph 3, 4-DiF-Ph 2, 3-DiF-Ph 3-F-Ph 4-F-Ph 2-Pyrrolyl 2-Furyl 3-Furyl 2-Thienyl 3-Thienyl

R

>1000 >1000 >1000 828 >1000 231 27 35 191 329

GSK-3α, IC50 nM

>1000 >1000 >1000 >1000 >1000 497 631 >1000 341 484

CDK-2, IC50 nM

STRUCTURE-BASED DESIGN OF 6-[HET]ARYL PYRAZOLOPYRIDINES

297

298

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

TABLE 15.16 Inhibition of hGSK-3a and CDK-2 by 6-heteroaryl pyrazolopyridines

R

O

H N

2

R

3

N R R2

R3

GSK-3α, IC50 nM

CDK-2, IC50 nM

Ph 4-HO-Ph 2-Thienyl 2-Thienyl 2-Furyl 2-Furyl 2-Thiazoyl 2-Thiazoyl 2-Thienyl 2-Furyl

Br Br Br Br H Br Br Br Br Br

cyPr cyPr cyPr cyPent cyPr cyPr cyPr cyPent CH 2-4-Piperidine-N-Et (±)-Pyrrolidine-N-Bn

67 <2 39 7 93 4 99 8 12 14

>10,000 5 639 1,699 >10,000 >10,000 1,007 >10,000 >10,000 >10,000

CDK2/cyclin A

As is evident from cross screening of 92 against a panel of kinases, and in contrast to the related 5-arylpyrazolopyridine and 5-arylpyrazolopyridazine series, a basic side chain is not required in order to obtain exquisite selectivity proﬁ les (Table 15.17).

15.4 CYTOCHROME P450 AND IN VITRO MICROSOMAL CLEARANCE DATA FOR 5-ARYL PYRAZOLOPYRID[AZ]INES AND 6-[HET]ARYL PYRAZOLOPYRIDINES In order to understand the potential developability liabilities, a number of diverse compounds from the various inhibitor classes were assessed for inhi-

GSK-3β

7 10 1 31 83

SGK

4

SAPK4

SAPK3

0 18 13 9

SAPK2b

1

SAPK2a

0

ROKa

0

PRAK

PKCA

19 3 16 8

PKBa

2

PKA

MEK1

6

PHOS.K

MAPKAP-K2

9

PDK1

RSK-2

8

P70S6K

MAPK

2

MSK1

LCK

92 5 13 8

Selectivity proﬁ ling of pyrazolopyridine 92

JNK

CKII

Chk1

AMPK

Compound

TABLE 15.17

N H

N

R1

Compound 57 52 89 90 91 92 93 94 95 96

1

IN VIVO EFFICACY DATA

299

bition of cytochrome P450s (CYP450) and in vitro microsomal clearance measurements (Table 15.18). In the 5-aryl pyrazolopyrid[az]ine series, rat microsomal clearance appeared to correlate with the overall lipophilicity of the compounds as reducing the cLogP by variation of the C-3 aryl group, incorporation of a basic center, or variation of the nitrogen atoms in the central core, all had a dramatic effect on lowering the clearance (cf. 11, 13, and 20 with 22, 28, 29, and 36). Interestingly in some cases cross species differences between rat and human microsomal clearance were also observed (e.g., 13). In general, the CYP450 proﬁ les for the C-5 phenyl analogues were encouraging (e.g., 11, 13). Replacement of the C-5 phenyl substituent with either the potency-enhancing 2,3-diF phenyl or a 3-pyridyl group gave mixed results. Introduction of the 2,3-diF phenyl ring led to a decrease in IC50s against 2C9 and 3A4, presumably due to an increase in lipophilicity (cf. 11, 20), while introduction of a 3-pyridyl group at C-5 led to a dramatic reduction in CYP1A2 IC50 (cf. 11, 22). Thankfully, incorporation of a basic amine group onto the amide side chain led not only to an improved solubility, selectivity, and microsomal clearance proﬁ le but also afforded a dramatic improvement in the overall P450 proﬁle (cf. 28 with 32 and 36). A number of compounds from the 6-aryl-pyrazolopyridine series were also assessed for inhibition of cytochrome P450s and in vitro clearance measurements (Table 15.19). Rat microsomal clearance appeared to be generally low except for analogue 92, and again, like the 5-aryl-pyrazolopyidine and the 5aryl-pyrazolopyidazine series, there appeared to be a reasonable correlation between rat and human microsomal clearance. Also noteworthy is the excellent P450 proﬁ les that appear to be in part due to the core nucleus, as changes to the C-6 substituent or the amide moiety have little effect on the overall P450 proﬁ les.

15.5 IN VIVO EFFICACY DATA FOR A SELECTIVE, ORALLY BIOAVAILABLE GSK-3 INHIBITOR Having identiﬁed compound 32 as the ﬁ rst potent and selective water-soluble GSK-3 inhibitor, the compound was proﬁ led in vivo in order to ascertain whether it was suitable as a tool compound for target validation studies. Although compound 32 was determined to have a low to moderate clearance in the rat, it did display low oral bioavailability, possibly due to poor oral absorption. Compound 32 did, however, possess an overall proﬁ le that was acceptable for target validation studies. Previously it had been demonstrated in the literature that selective small molecule inhibitors of GSK-3 are able to stimulate glycogen synthase in vitro and hence have potential in the treatment of non-insulin-dependent diabetes mellitus [12]. It was found that compound 32 was also able to activate glycogen synthase in human Chang cells (EC50 0.7 μM), and so the compound was

Y

C C C

C N N N N

11 13 20

22 28 29 32 36

N N C N N

N N N

X

Ph Ph 2,3 Dif-Ph 3-Pyridyl Ph Ph Ph Ph

R1

Bu Bu Bu (CH 2) 3NMe2 4-PiperidineN-Me

Bu cPentyl Bu

R2

1

R N H

N

5.6 2 12 <0.5 2.1 5.4 <0.5 3.3

<0.5 6.3 14 0.8 <0.5

Human

Microsomal Clearance (ml/min/g) Rat

Y

2

R

O

11 >50 22

X

H N

Cytochrome P450 inhibition and microsomal clearance data

Compound

TABLE 15.18

<0.1 2.7 4.2 54 26

5.4 5.2 8.9

1A2

72 39 39 >100 >100

25 38 15

2C19

68 6.8 33 >100 52

23 67 5.7

2C9

21 >100 >100 >100 >10

67 >100 56

2D6

Human P450 IC50 (μM)

11 31 >100 >100 9.7

29 28 3

3A4

300 3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

61 64 92 95 96

N

N

Compound

32

TABLE 15.19

N H

N

H N

O

Ph 4-HO-Ph 2-Furyl 2-Thienyl 2-Furyl

R1

NMe2

N

N H

N

R

O 3

3.9 0.9 23 1.7 5.9

Rat 2.2 <0.5 5.8 2.1 8.8

Human

Microsomal Clearance (ml/min/g)

GSK-3α, IC50 22 nM CLb 32 ml/min/kg Vss 1.5 L/kg T1/2 (i.v.) 1.3 h Cmax 0.100 μM Fpo 14%

(±)-Pyrrolidine-N-Bn 4-Piperidine-N-Me cyPr CH 2-4-Piperidine-N-Et (±)-Pyrrolidine-N-Bn

R2

R

1

Br

H N

Cytochrome P450 inhibition and microsomal clearance data

13 99 37 32 25

1A2

17 nd >100 50 >100

2C19

29 nd >100 >100 >100

2C9

30 nd >100 68 >100

2D6

100 >100 >100 >100 >100

3A4

Human P450 Isoform IC50 (μM)

IN VIVO EFFICACY DATA

301

302

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS

Effect of 32 on oral glucose tolerance in the ob/ob mouse 30 Control 300 μmol/kg

Blood glucose conc (mmol/l)

25

100 μmol/kg 30 μmol/kg

20

errors = sd's 15

10

5

0 –90

–45

Figure 15.7

0 45 Time (mins)

90

135

Oral efﬁcacy data for compound 32 in obese mice.

proﬁ led further in obese mice and diabetic rats. Oral administration of 32 to 18-hour fasted ob/ob mice 1-hour before an oral glucose load, resulted in statistically signiﬁcant lowering of blood glucose in all doses tested (−41%**, −27%**, −17%* @ 300, 100, and 30 μmol/kg, respectively, *P < 0.05, **P < 0.01; 300 μmol/kg = 108 mg/kg) (Fig. 15.7). An initial study with 32 in the 18-hour fasted ZDF rat to determine the effect on blood glucose levels over 6 hours (no OGTT) showed signiﬁcant reduction in AUC compared with control, although in this case there was no dose dependency and no efﬁcacy at 30 μmol/kg (−30%**, −29% ** @ 300 and 100 μmol/kg, respectively, **P < 0.01; Fig. 15.8). Interestingly, repeating the experiment at 30 μmol/kg and 100 μmol/kg, but sacriﬁcing the animals at 3-hour postdose in order to measure glycogen synthase (GS) activity and glycogen levels in muscle and liver, produced some noteworthy ﬁ ndings. In both the dosage groups there was a statistically signiﬁcant elevation of GS activity in both liver and muscle (Fig. 15.9).

303

IN VIVO EFFICACY DATA 16.00 Control

Blood glucose conc (mmol/l)

14.00

300 μmol/kg

12.00

100 μmol/kg errors = sd's

10.00 8.00 6.00 4.00 2.00 0.00 –60

0

Figure 15.8

60

120 180 Time (mins)

240

300

Effect of 32 on blood glucose concentration in the ZDF rat.

Mean +/- se

Muscle

Liver

G-6-P ratio

360

0.6

0.6

0.5

0.5

0.4

***

0.3

***

*** ***

0.4 0.3

0.2

0.2

0.1

0.1 0

0 Control

Figure 15.9

30

100

Control

30

100

Effects of 32 on glycogen synthase activity in the ZDF rat.

304

3-AMINO PYRAZOLES AS POTENT AND SELECTIVE GSK-3 INHIBITORS Muscle (GP)

Liver

* mg glycogen / g muscle

40 mg glycogen/ g liver

2.5

20

2 1.5 1 0.5 0

0 Control

30

R R

2a

R

2

R

b

H N N H

100

N

30

100

c

Z Z

Control

2

R

1

Q

R

X R

1

1

Bebbington, US2005038023

N

O R

2

4

N N R3

Vertex, WO2004072029

Figure 15.10 Effects of 32 on liver and muscle glucogen concentration in the ZDF rat.

Tissue glycogen was elevated only in liver at the higher dose level (Fig. 15.10). These data might suggest that GS activity could be a more sensitive pharmacodynamic measure of compound effects than blood glucose lowering.

15.6 EMERGING TEMPLATES CONTAINING A 3-AMINO PYRAZOLE SCAFFOLD The use of a functionalized 3-amino pyrazole scaffold continues to provide a way into identifying novel kinase scaffolds. Recent patents from both Bebbington Vertex suggest further modiﬁcations can afford compounds with activity against GSK-3 beta, although nothing has been reported about the selectivity or in vivo properties of these analogues.

REFERENCES

305

REFERENCES 1. Aoki M., Iwamoto-Sugai M., Sugiura I., Sasaki C., Hasegawa T., Okumura C., Sugio S., Kohno T., Matsuzaki T. (2000). Acta Crystallogr., Biol. Crystallogr., D56, 1464. Bax B., Carter P.S., Lewis C., Guy A.R. Bridges A., Tanner R. Pettman G. Mannix C., Culbert A.A., Brown M.J.B., Smith D.G., Reith A.D. (2001). Structure (London), 9, 1143. 2. Furet P., Caravatti G., Lydon N., Priestle J., Sowadski J., Trinks U., Traxler, P. (1995). J. Comput. Aided Mol. Des., 9, 465. 3. Traxler P, Furet P. (1999). Pharmacol. Ther., 82, 195. 4. Witherington J., Bordas V., Garland S.L., Hickey D.M., Ife R.J., Liddle J., Saunders M., Smith, D.G. (2003). Bioorg. Med. Chem. Lett., 13, 1577. 5. Davies S.P, Reddy H., Caivano M., Cohen P. (2000). Biochem. J., 351, 95. 6. Witherington J., Bordas V., Haigh D., Hickey D.M., Ife R.J., Rawlings A.R., Slingsby B., Smith D.M., Ward R. (2003). Bioorg. Med. Chem. Lett., 13, 1581. 7. Duvadie R.K., Chong W.K.M., Li L., Chu S.S., Yang Y.M., Nonomiya J., Tucker K.D., Lewis C.T., Knighton D.R., Ferre R.A., Lundgren K., Koudriakova T., Escobar J., Minnick P.S., Huber A., Sisson W., Aust R.M., Verkhivker G.M., Schaffer L., Rose P.W. (1999). Abstracts. 218th ACS National Meeting, New Orleans. 8. Schulze-Gahmen U., De Bondt H.L., Kim S.-H. (1996). J. Med. Chem., 39, 4540. 9. Bowler A.N., Olesen P.H., Sorensen A.R., Hansen B.F., Worsaae H., Kurtzhals P. WO 0156567. 10. Witherington J., Bordas V., Gaiba A., Naylor A., Rawlings A.R., Slingsby B.P., Smith D.G., Takle A.K., Ward R.W. (2003). Bioorg. Med. Chem. Lett., 13, 3055. 11. Witherington J., Bordas V., Gaiba A., Naylor A., Rawlings A.D., Slingsby B.P., Smith D.G., Takle A.K., Ward R.W. (2003). Bioorg. Med. Chem. Lett., 13, 3059. 12. Coghlan M.P., Culbert A.A., Cross D.A.E., Corcoran S.L., Yates J.W., Pearce N.J., Rausch O.L., Murphy G.J., Carter P.S., Cox L.R., Mills D., Brown M.J., Haigh D., Ward R.W., Smith D.G., Murray K.J., Reith, A.D., Holder J.C. (2000). Chem. Biol., 7, 793.

16 MARINE COMPOUNDS AS A NEW SOURCE FOR GLYCOGEN SYNTHASE KINASE 3 INHIBITORS Diana Alonso and Ana Martínez NeuroPharma, Avda. de la Industria 52, Tres Cantos, Madrid

Contents 16.1 Introduction 16.2 Hymenialdisine and Pyrrole Alkaloids 16.2.1 Structural Analogues of HD 16.2.2 Hydrazones and Amides Related to HD 16.3 Indole and bis-Indole Alkaloids 16.3.1 Meridianins 16.3.2 Indirubins 16.4 Manzamines 16.5 Palinurin and Furanosesquiterpenoids 16.6 Genisteine and Isoﬂavones 16.7 Concluding Remarks

16.1

INTRODUCTION

Glycogen synthase kinase 3 (GSK-3) is a serine-threonine kinase ubiquitously expressed and involved in the regulation of many cell functions [1]. GSK-3 was originally identiﬁed as one of the ﬁve protein kinases that phosphorylate Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

307

308

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

glycogen synthase being implicated in type 2 diabetes [2]. GSK-3 is also known to phosphorylate the microtubule associated protein Tau in mammalian cells [3]. This hyperphosphorylation is an early event in neurodegenerative conditions such as Alzheimer’s disease (AD). GSK-3 emerged in the twenty-ﬁ rst century as one of the most attractive therapeutic targets for the development of selective inhibitors as new promising drugs for severe unmet pathologies such as diabetes type 2, bipolar disorders, stroke, AD, and different Tau pathologies such as Pick’s disease, supranuclear palsy, and frontotemporal dementia [4]. A number of recent publications have emerged describing structurally diverse molecules that inhibit GSK-3 such as pyridyl-oxadiazoles [5], maleimides [6], thiadiazolidindiones, and pyrazolopyrimidines. The two latter families are widely described in this volume. Current advances in the search for GSK-3 inhibitors have been recently reviewed [7–9]. It is interesting to note the prominent role that marine invertebrates have played in the generation of novel GSK-3 inhibitors, including hymenialdisine, meridianines, and indirubines isolated from several diverse sponges, ascidians, and gastropod molluscs, respectively. Indeed, the marine environment represents an enormous resource for the discovery of potential therapeutic agents [10,11], and the search for GSK-3 inhibitors is one of the successful areas. Furanosesquiterpene palinurine, the complex alkaloid manzamine A, and the isoﬂavone genisteine produced by a marine microorganism are some of the most recent examples. All these compounds will be reviewed in this chapter.

16.2

HYMENIALDISINE AND PYRROLE ALKALOIDS

Hymenialdisine 1 (HD) belongs to a family of marine-sponge-derived natural products that contain both bromopyrrole and guanidine groups [12]. It was originally isolated from sponges belonging to the Agelasidae, Axinellidae, and Halichondridae families, and its structure was established using X-ray crystallography [13]. Moreover these sponges contain also a great variety of substances, which are clearly metabolically related to HD (Fig. 16.1). HD is a kinase inhibitor with nanomolar activity against GSK-3β, CDKs, MEK1, and CK1 and micromolar activity against Chk1 [14–16]. In addition HD has the ability to inhibit GSK-3β in vivo and also blocks the in vivo phosphorylation of the microtubule-binding protein Tau at sites that are hyperphosphorilated by GSK-3β in AD. As observed with other GSK-3β inhibitors, HD and derivatives thereof act in competition with ATP [17]. HD and some of its metabolically related compounds are depicted in Figure 16.1. HD is the most active compound derived from them, followed by axinohydantoin 2 and dibromocantharelline 3. Both compounds display a signiﬁcant inhibitory effect toward GSK-3β with an IC50 of 3 μM. The activities of HD as a potentially antiproliferative, anti-inﬂammatory, and antineurodegenerative agent have been investigated in several models

309

HYMENIALDISINE AND PYRROLE ALKALOIDS

O

H2N

N

HN Br

O

O

N H

NH O

O

Axinohydantoin 2 IC50=3 μM

Hymenialdisine 1 IC50=10 nM

N H

NH

NH2

N

Br Br

Br N H

NH

HN

NH N

O

Dibromocantharelline 3 IC50 =3 μM

CH3 HO N O

Br

NH

Br Br

NH O

N H

Br N

Br

NH

Sceptrin 7 (-)

NH

N H

O

H N

NH N

HN

NH2

Br Br Br

N

NH2 NH

O Clathrodin 6 IC50=10 μM

Br

NH2

NH

O

N

Oroidin 5 IC50 =20 μM

Agelastatin A 4 IC50=12 μM

N H O NH

H N

NH2

H2N

N

HN NH N H O NH NH O

N N H

NH2

Dibromoageliferin 8 IC50=11 μM

Figure 16.1 Hymenialdisine and related metabolically compounds with their inhibition on GSK-3.

[18]. For example, it has been reported that HD inhibits NFκB activity through an alternative mechanism to the inhibition of protein kinase C or IκB phosphorylation [19,20]. The possibility that CDKs, GSK-3β, and CK1, or another unknown kinase target of HD, are involved in NFκB activation and in adjuvant-induced arthritis deserves further investigation. Total synthesis of HD has been reported by two independent research groups [21,22]. Moreover an extensive analogues program has been described, enabling the ﬁ rst structure-activity relationship study [23]. Crystallographical data of the HD complex with CDK2 [17] led to the rational development of

310

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

new analogues with increased potency and selectivity over GSK-3, CDK5, and CDK1. 16.2.1

Structural Analogues of HD

When the inhibitory activity of the analogues against GSK-3 was examined, we can observe that most of the new molecules synthesized are potent inhibitors (Table 16.1). As shown, non-, mono-, or dihalogenation at the 2- and 3position on the pyrrole ring has little effect on the activity of these compounds against GSK-3β (compounds 1 versus 9–12). However, substitution in the azepine and glicocyamidine core reduce the inhibitory potency (compound 9 versus 13, 14, and 15). Moreover an additional ring fused to the central pyrrole-azepinone heterocycle led to derivatives that generally exhibit similar activity compared to the corresponding pyrrole derivatives (compounds 16– 26). As regards selectivity, it was observed that GSK-3 and CDK5 inhibition remains at the same rate of potency (compounds 9–15), showing a property common to most cyclin-dependent kinase inhibitors. The most important achievement is that unlike HD, these analogues present different kinases selectivities, and interestingly the 7-bromoindole analogue 19, is, for instance, 10 times more active against GSK-3β than against CDKs. 16.2.2

Hydrazones and Amides Related to HD

In order to replace glycocyanamide ring of HD, several hydrazones were introduced in the dibromopyrroloazepinone skeleton. In all cases hydrazone derivatives are less potent against GSK-3β and CDK5/p25 (Table 16.2) [23]. In addition, derivatives obtained by substitution of bromopyrrole ring (compounds 31–34), show reduced potency against GSK-3β. However, deeper analysis could prove that favorable selectivity toward GSK-3β as against CDK5/p25 can be achieved. For example, hydrazone 28 is an inhibitor of GSK-3β, while it is devoid of any activity against CDK5/p25. In conclusion, by chemical structure modiﬁcations, several compounds related to HD have been obtained with great selectivity toward GSK-3. The interesting in vitro inhibitory proﬁ le of the bromo-indole 19 makes this compound a promising lead for the future treatment of diseases in which the inhibition of GSK-3β plays an important role. Currently different research groups are actively working in this area with different synthetic programs [24].

16.3

INDOLE AND BIS-INDOLE ALKALOIDS

Marine invertebrates are a proliﬁc source of indole alkaloids [25], especially from tunicates and sponges, among which are meridianins and indirubins derivatives as GSK-3 inhibitors.

N H

HN

O

R2

R4HN

R1

HN

N

Compound

R1

R2

R4HN

Compound

O

N H

O

R3

O

NH

R3

16 17 18 19 20 21 22 23 24 25 26

1 9 10 11 12 13 14 15

H H H H H H H NO2 NH 2 H H

R1

Br Br H H Cl Br Br Br

R1

H F Cl Br SO2Me NO2 NH 2 H H Br Br

R2

H Br H Br Cl Br Br Br

R2

H H H H H H H H H Me H

R3

H H H H H Me H H

R3

H H H H H H H H H H Ac

R4

H H H H H H Ac Et

R4

64 52 51 47 120 710 400 48 163 175 2,700

GSK-3β

73 51 111 90 47 268 363 1,388

GSK-3β

800 550 2,300 1,000 890 3,200 1,600 1,200 1,600 500 ≥10

CDK1/cyclinB

IC50 /nM

70 530 250 1,500 220 500 1,500 >10,000

CDK1/cyclinB

IC50 /nM

Close analogues of HD, and their biological activity against GSK-3b, CDK5/p25, and CDK1/cyclin B

NH

N

TABLE 16.1

177 177 191 213 1,240 713 681 109 187 1,010 5,110

CDK5/p25

37 56 112 79 62 251 533 9,120

CDK5/p25

INDOLE AND BIS-INDOLE ALKALOIDS

311

312

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

TABLE 16.2 HD analogues with the replacement of the glycocyamidine ring and their IC 50 in mM against GSK-3b, CDK5/p25, and CDK1/cyclin B CO 2H

NO2

N

CO2H NH

NH

N H

NH O

Compound GSK-3b CDK5/p25 CDK1/cyclinB

Br

N H

NH O

NH

28

29

30

82.4 >33 ND

7.4 >100 ND

4.28 10.4 ND

16.6 3.8 ND

N

N NH

N

Cl

N NH

F

NH O

N H

NH O

NH

N

N

Ph

GSK-3b CDK5/p25 CDK1/cyclinB

NH O

27

N

Compound

N H

O

NH

N H

N

Br

Br

N H

N

Cl

NH

N

Br

Br

Br

16.3.1

NH

N

Br

N

Br

N H

NH O

H2N

N H

NH O

31

32

33

34

6.99 6.91 ND

9.13 23.2 ND

0.171 0.081 1.3

5.95 22.5 ND

Meridianins

Meridianins are brominated 3-(2-aminopyrimidine)-indoles that are isolated from the tunicate Aplidium meridianum [26] collected near the South Georgia Islands. As part of an ongoing research program into ﬁ nding molecules involved in cell cycle control and neuronal functions [27], the ability of meridianins to inhibit several protein kinases was discovered [28,29]. Kinase activities were determined in different assays using a 15 μM ATP concentration, and in the presence of increasing meridianin concentration. When the inhibitory activity of the meridianins against GSK-3β was determined (Table 16.3), it is clear that a bromine substitution in position 6 and an hydroxyl in position 4 of the indole ring (compound 36) provide the best inhibitory activity. Further these compounds have speciﬁcity against other kinases, such as CDK5 and CDK1. The lack of substituents in the indole ring leads to compounds inactive against GSK-3 (derivative 41). The indole position where the 2-aminopyrimidine is attached is crucial for GSK-3 inhibition, and the isomeridianins 42–43 are completely inactive (Table 16.4).

313

INDOLE AND BIS-INDOLE ALKALOIDS

TABLE 16.3 Meridianins and their IC 50 in mM against GSK-3b, CDK5/p25, and CDK1/cyclin B

H2N R1

N

N

R2 R3 R4 Compound

35 36 37 38 39 40 41

R1

OH OH H H OH H H

R2

R3

H H Br H H Br H

H Br H Br H Br H

N H

R4

IC50 /μM

H H H H Br H H

GSK-3β

CDK1/cyclinB

CDK5/p25

1.30 0.50 2.00 2.50 2.50 2.00 350.00

2.50 1.50 3.00 13.00 0.18 20.00 150.00

3 1 6 5.5 0.15 20 140

TABLE 16.4 Isomeridianins and their IC 50 in mM against GSK-3b, CDK5/p25, and CDK1/cyclin B

R N N H Compound

R

N NH2 IC50 /μM

GSK-3β CDK1/cyclinB CDK5/p25 42 43

Br H

>1000 420.00

160.00 140.00

300 130

More recently several groups have reported different syntheses of natural meridianins and their derivatives based on different synthetic approaches. However, their GSK-3 activity evaluations have not been disclosed yet [30–32]. 16.3.2

Indirubins

Indirubins are derived from the spontaneous nonenzymatic dimerization of Indolxil and Isantin (Fig. 16.2). These two colorless precursors are found,

314

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

O

OH N H Indoxyl

Figure 16.2

O N H

O

N H Indirubin

N H Isantin

O

Indirubin and indirubins’ precursors.

either free or conjugated to carbohydrates, in over 200 species of indigoproducing plants [33]. Indirubins, along with indigo (Fig. 16.2), are also produced by several bacterial strains [34] and by a number of gastropods molluscs [35]. During the course of an intensive effort to prepare synthetic indigo to help the rapidly expanding of dye industries, Adolf von Bayer achieved the ﬁ rst total synthesis of indirubin in 1870 [36]. More than 100 years later, in 1979, indirubin was reported in a publication to be the active constituent of a traditional Chinese antileukemia remedy. This ﬁ nding prompted further investigation into the pharmacological properties of indirubins, thereby discovering the ATP-competitive inhibition of CDKs [37,38], a key target in the ongoing search for novel antitumor therapies [39–41].. The kinase selectivity study performed with these compounds led to the discovery that GSK-3 is also an excellent target for indirubins. In Table 16.5 the activities of a large range of indirubin derivatives against GSK-3β and CDKs is shown. As regards GSK-3 inhibition, the SAR of these derivatives discloses that the compounds that have a carbonyl group in position C3′ show, in general, good inhibition against GSK-3β. Moreover compounds 47–50 are 10 times more potent against this kinase than as against CDKs. The same selectivity is observed in compounds 55 and 56, when an oxime group replaces the carbonyl group. In contrast, N-1 substitutions give poorly active derivatives in all kinases, as is shown for compounds 58 and 59. This analysis shows that the efﬁcacy of indirubins toward CDK1 and CDK5 is closely related, whereas the efﬁcacy toward GSK-3β differs from that of CDKs. This probably reﬂects the closer evolutionary proximity between CDK1 and CDK5 compared with that between GSK-3β and CDKs [42]. In the series of analogues, depicted in Table 16.5, it is worth mentioning that the compound most active on GSK-3β, is 5-iodo-indirubin-3′oxime 56, being 100 times more potent that indirubin 44, while the most GSK-3β-selective compound is 5,5′-dibromoindirubin 53, being 1000 times more active against GSK-3β than as against CDKs. Additionally indirubin-3′-monooxime 55 inhibits Tau phosphorylation in vitro and in vivo at AD-speciﬁc site [38]. Indirubins may thus constitute lead compounds in the study and treatment of neurodegenerative disorders involving abnormal phosphorylation of Tau.

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

Compound

TABLE 16.5

H H H H H H H H H H H H H H CH 3 Phenyl H H H H

1

C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O C=O =N—OH =N—OH C=O C=O C=O =N—OH C=O C=O C=O

3′

7´

4´

H

2´

O

N 1´

3´ O

H I Br Cl F CH 3 NO2 SO3H H Br SO3H H I H H H SO3Na SO2NH 2 SO2 —N(CH 3) 2 SO2 —N—C 2H 5 —OH

5

6´

5´

N1 H

H H H H H H H H Br Br Br H H H H H H H H H

5′

2

3

H H H H H H H H H H H H H I H H H H H H

6

7

4

6

5

H H H H H H H H H H H H H H H H H H H H

6′

0.60 0.068 0.55 0.050 0.078 0.062 0.042 0.28 0.35 0.25 4 0.022 0.009 0.13 >1000 200 0.080 0.040 0.18 0.033

GSK3β

IC50 /μM

10 0.35 0.23 0.28 0.35 0.28 0.25 0.050 0.51 600 0.080 0.18 0.025 0.80 >1000 500 0.005 0.11 0.10 0.065

5.5 0.2 0.25 0.23 0.75 0.21 0.38 0.065 4 200 0.075 0.1 0.02 1.5 >1000 800 0.007 0.075 0.06 0.05

CDK1/cyclinB CDK5/p25

Indirubins’ derivatives and their IC 50 in μM against GSK-3b, CDK5/p25, and CDK1/cyclin B

INDOLE AND BIS-INDOLE ALKALOIDS

315

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

Compound

TABLE 16.5

H H H H H H H H CH 3 CH 3 CH 3 H H H H H

1

3′

C=O C=O C=O C=O C=O =N—OH =N—OH =N—OH =N—OH C=O =N—OH =N—O—CH 3 =N—O—Ac =N—O—Ac =N—O—CH 3 C=O

Continued

7´

4´

N 1´ H O

2´

3´ O

SO2 —N—(C 2H 5OH) 2 SO2 —NHCH 3 H H H H H H H H H H H H H H

5

6´

5´

N1 H

H H H H H H H H H H H H H H H H

5′

2

3

H H H Br Br H Br Br H Br Br Br Br H H Cl

6

7

4

6

5

H H Br H Br Br H Br H H H H H H H H

6′

0.40 0.11 22.00 0.045 4.50 0.34 0.005 0.12 >100.0 >100.0 44.00 0.03 0.01 0.200 0.150 0.140

GSK3β

0.15 0.080 >100.0 90.00 100.00 3.00 0.32 17.00 73.00 80.00 55.00 3.40 63.00 1.200 1.000 >100

0.15 0.02 >100.0 53 >100.0 1.2 0.083 1.3 >100.0 >100.0 >100.0 2.2 2.4 0.7 0.4 >100

CDK1/cyclinB CDK5/p25

IC50 /μM

316 MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

H H H H H H H H H H CH 3 CH 3 H H H H H H H H H

=N—OH =N—O—Ac =N—OH =N—O—Ac C=O =N—OH =N—O—Ac C=O =N—OH =N—O—Ac C=O =N—OH C=O =N—OH =N—O—Ac C=O =N—OH =N—O—Ac C=O =N—OH =N—OH

H H H H H H H H H H H H CH 3 CH 3 CH 3 Cl Cl Cl NO2 NO2 NO2

H H H H H H H H H H H H H H H H H H H H H

Cl Cl I I CH = CH 2 CH = CH 2 CH = CH 2 F F F H H Br Br Br Cl Cl Cl Br Br Br

H H H H H H H H H H H H H H H H H H H H H

0.020 0.017 0.010 0.013 0.240 0.060 0.065 0.650 0.130 0.090 >100 >100 0.025 0.006 0.007 0.030 0.004 0.004 0.100 0.007 0.006

0.650 30.000 1.300 2.200 4.200 1.200 1.600 1.500 0.320 0.600 >100 73.000 30.000 0.300 31.000 45.000 0.140 30.000 >100 12.000 11.000

0.1 0.2 0.3 1.3 2.4 0.42 0.4 1 0.15 0.3 >100 >100 60 0.006 30 60 0.06 0.1 >100 0.15 31

INDOLE AND BIS-INDOLE ALKALOIDS

317

318

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

In an effort to identify new kinase inhibitors with increased potency and selectivity, natural indirubins produced by the Mediterranean molusk Hexaplex trunculus were investigated. Several bromo-substituted indirubins (66–76) were identiﬁed, the most acitive being a GSK-3β inhibitor, 6-bromoindirubin 67. These synthetic 3’-oxime derivatives 69–71 constitute unique cell-permeable compounds with enhanced solubility and kinase inhibition [38]. From these, 6-bromoindirubin-3′-oxime 70 called BIO is a selective inhibitor of GSK-3 that mantains pluripotency in human and mouse embryonic stem cells, thus pointing to a practical application in regenerative medicine [43]. The 1-methyl derivatives 72–74 were also synthesized, but no relevant activity against GSK-3 was found. In fact methylation in position N1 led to indirubins 72 and 73, derivatives devoid of any activity as kinase inhibitors. Finally, 3′-oxime in compound 70 was also replaced by 3′-methoxime 75 or 3′-acetoxime 76. In both cases the activity against GSK-3 decreased, but the selectivity against CDKs was enhanced 100-fold. Moreover, because of the good results obtained as GSK-3 inhibitors by these analogues, the compounds 55, 70, 75, and 76 were tested on a series of 20 puriﬁed protein kinases in the presence of 15 μM ATP. The results of these assays conﬁ rm the strong selectivity of 6-bromo-substituted indirubins for GSK-3 (Table 16.6). The high selectivity for GSK-3 of 6-bromoindirubin 66 and BIO prompted research into the role of the substituent at position 6. With

TABLE 16.6

Selectivity of indirubins against 20 kinases

Protein Kinases GSK3α/β CDK1/cyclinB CDK2/cyclinA CDK4/cyclinD1 CDK5/p35 Erk1 Erk2 MAPKK Protein kinase C α Protein kinase C β1 Protein kinase C β2 Protein kinase C γ Protein kinase C δ Protein kinase C ε Protein kinase C η Protein kinase C ζ cAMP-dependent PK cGMP-dependent PK Casein kinase 2 Insulin receptor Tyr kinase

55

70

75

0.022 0.18 0.44 3.33 0.10 >100 >100 >100 27 4 20 8.40 >100 20 52 >100 6.3 9 12 11

0.005 0.32 0.30 10 0.08 >10 10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

0.030 3.40 15 >10 2.20 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 9 >10

76 0.010 63 4.3 >10 2.4 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

319

MANZAMINES

this purpose, a large number of derivatives have been synthesized [44]. Recent crystallographic data of CDK2 [39], CDK5, and GSK-3 [44] in complex with several indirubins has provided valuable information on the active site interactions, with each kinase. These data were used in the rational design of a new generation of compounds [45]. Substitution at position 6 turned out to be crucial for the selectivity, while substitution at 3′was found to be important for the binding afﬁ nity to each kinase. It has been noticed that the conversion of the C3′carbonyl to an oxime improves inhibitory potency against GSK-3 (compounds 80, 81, 85, 86, 88, 89, 93, 94, 96, 97, 99, and 100). In conclusion, indirubins constitute a promising molecular scaffold from which selective molecules active on GSK-3 and CDKs are able to be synthetized with some therapeutically useful properties against some diseases where these kinases are implicated. Recently Schering AG has reported new indirubin derivatives with improved solubility properties [46]. 16.4

MANZAMINES

Manzamines are complex β-carboline alkaloids isolated from Indo-Paciﬁc sponges and characterized as having an intricate and novel polycyclic system. In 1986, Higa and co-workers ﬁ rst reported manzamine A 101 from the Okinawan sponge of the genus Haliclon [47]. To date, 16 species belonging to 8 families of marine sponges, including Acanthostrongylophora, have been conﬁ rmed to yield β-carboline manzamine and manzamine-related alkaloids [48]. The occurrence of manzamine alkaloids in a diversity of unrelated sponges has led to speculation of a possible microbial origin for the biosynthesis of these compounds [49]. Following a discovery program of GSK-3 inhibitors from marine sources, it was found that manzamine A inhibits human GSK-3β in vitro more than 70% at 25 μM [50]. In order to identify the pharmacophore responsible for this new enzymatic inhibition, the potential GSK-3 inhibition of carboline 102 and ircinal A 103, which can be considered the chemical precursors of manzamine A, were tested. Both moieties are inactive in their ability to bind to GSK-3 (Fig. 16.3), indicating the entire manzamine molecule is responsible for this activity. H

O

OH

N N

N N H 103

102

Figure 16.3 Ircinal and carboline structures.

320

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

Next, the structure-activity relationship (SAR) of this new chemical class was investigated, testing in an scintillation assay several manzamines analogues isolated from different sponges or prepared through semisynthetic modiﬁcation. The effect of some substituents on the carboline moiety was examined as well as the effect of some changes in the aliphatic heterocyclic system (Table 16.7). Replacement of hydrogen at carboline 6 or 8 position with OH, OMe, OEt, or OTos groups is well tolerated by the enzyme, and compounds 104–108 are equipotent to manzamine A. However, substitution of nitrogen 9 of the carboline heterocycle results in noticeable changes to GSK-3β inhibition. Therefore small groups such as Me or Et are well tolerated by the enzyme (compounds 107 and 108), while the larger ones such as iPr, (CH 2)11CH 3, or ButOCOMe led to inactive derivatives (compounds 109, 110, and 111). Conformational restriction in the aliphatic heterocyclic system of manzamines elicits a signiﬁcant effect on GSK-3 inhibition. Thus the absence of a double bond between positions 15 and 16 results in inactive derivatives (compounds 113 and 116), while the lack of Δ32 unsaturation allows slight structural changes. While compound 112, with an epoxy function in the cyclooctane ring, maintains the same GSK-3 inhibition of manzamine A, derivatives 114 and 115, with a carbonyl group in position 31, are less potent than manzamine A 101 and compound 104, respectively. However, if the hydroxyl group of derivative 115 is substituted with different bulky moieties (compounds 117– 119), an increase in GSK-3 inhibition is observed. This fact points to the possibility of a hydrophilic pocket in the GSK-3 enzyme where the phenyl ring of the carboline heterocycle is allocated. Moreover, if conformational restriction is increased in the aliphatic part of manzamines, the inhibition of the enzyme is sustained (compounds 120 and 121). To further assess the potential of manzamine A in the treatment of AD, its ability to inhibit several different kinases (GSK-3β, GSK-3α, CDK-1, PKA, MAPK, and CDK-5) (Table 16.8) and decrease the hyperphosphorylation of Tau protein mediated by GSK-3 in human neuroblastoma cell cultures was investigated. Manzamine A speciﬁcally inhibits GSK-3β and CDK-5 with an IC50 of 10 and 1.5 μM, respectively, with the two key players in the hyperphosphorylation of Tau protein in AD [51] being inactive in the others kinases tested. In addition, treatment of SH-SY5Y cell culture with manzamine A at different concentrations (5, 15, and 50 μM) resulted in a decrease of Tau phosphorylation quantiﬁed by measuring with a speciﬁc ELISA sandwich method, the phosphorylation of the epitope in Ser396, which is phosphorylated speciﬁcally by GSK-3. Cell survival was determined in parallel by measuring LDH release. This result is a demonstration of its ability to enter cells and interfere with Tau pathology. Manzamine A constitutes a promising scaffold from which more potent and selective GSK-3 inhibitors could be designed as potential therapeutic agents for the treatment of diseases mediated by GSK-3 such as AD and further validates the marine environment as a rich source for new therapeutic agents for neurodegenerative diseases.

109

108

107

106

105

104

Compound

TABLE 16.7

N

N

N

N

N

N

R

OiPr

OEt

OMe

OTos

H

OH

R1

H

H

H

H

OH

H

R2

N

i

Pr

Et

Me

H

H

H

X´ N

R3

R

X Y

N

GSK-3β inhibition by manzamine analogues at 25 mM

R3 Y´

N R1

R2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

X-Y

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

X′-Y′

—

78.0

72.4

80.4

74.3

86.7

GSK-3β

MANZAMINES

321

115

114

113

112

111

110

Compound

TABLE 16.7

O

O

N

N

N

N O

N

N

R

Continued

OH

H

OH

H

H

H

R1

H

H

H

H

H

H

R2

N

X´ N

R3 Y´

N

H

H

H

H

ButOCOMe

(CH 2)11CH 3

R3

R

X Y

N R1

R2

CH 2 = CH 2

CH 2 = CH 2

(CH 2) 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

X-Y

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

X′-Y′

30.0

53.6

—

84.5

—

—

GSK-3β

322 MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

121

120

119

118

117

116

O

O

O

N

N

N

N

N

N

OCOMe

H

O OS O

O OS O

Br

H

H

H

H

N

Me

Cl

H NHCOMe

S

O S O

O

H

H

H

H

H

H

H

H

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

CH 2 = CH 2

(CH 2) 2

C = CH

C = CH

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

C(OH)—CH 2

79.1

71.2

63.0

76.7

78.8

—

MANZAMINES

323

324

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS

TABLE 16.8

Manzamine A inhibition proﬁ le on different kinases

N

N H OH

N N

101

GSK-3β IC50 (μM) 10

16.5

GSK-3α IC50 (μM)

CDK-5 IC50 (μM)

CDK-1 IC50 (μM)

PKA IC50 (μM)

MAPK IC50 (μM)

25

1.5

>25

>25

>25

PALINURIN AND FURANOSESQUITERPENOIDS

Very recently extracts and compounds obtained from the marine organism Ircinia sp., and, more particularly, the furanoterpenoids isolated from the mediterranean sponges Ircinia dendroides, Ircinia variabilis, and Ircinia oros have been claimed as inhibitors of GSK-3 [52]. Fractionation and puriﬁcation of active components from these extracts, guided by a GSK-3 inhibition assay, resulted in the isolation of furanosesquiterpenoids as new GSK-3 inhibitors with potential use as therapeutic agents. Palinurin [53] (122) and one unknown metabolite called tricantin (123) were mainly isolated (Fig. 16.4). The chemical structure elucidation of the compounds was performed on the basis of their spectrospical (IR, ES+, UV, 1HNMR, and 13C-NMR) data together with the results of bidimensional NMR experiments, both homonuclear, such as COSY and NOESY pulse sequences, and hetereonuclear experiments, such as HMQC and HMBC pulse sequences. Some furanosesquiterterpenes isolated from Ircinia sp. display a wide range of bioactivities, including cytotoxic activity [54], protein kinase inhibition [55], and antibiotic effects [56]. Moreover palinurin had been previously described as an anti-inﬂammatory and antibacterial compound [57], but the potent inhibition of GSK-3β of the isopropanolic extracts of the Ircinia sp. (90% of inhibition at 50 mg mL −1) and their active components, palinurin and tricantin, have been recently described. Kinetic analyses of isolated compounds were performed, and the results show that tricantin inhibits recom-

325

GENISTEINE AND ISOFLAVONES

HO

O

O 122

O

O 123

Figure 16.4

O

O

O O

Palinurin and tricantin chemical structures.

binant human GSK-3β with an IC50 value of 7.5 μM, whereas palinurin shows an IC50 value of 4.5 μM. In order to investigate the mechanism of inhibition of GSK-3β by palinurin, several kinetic experiments were performed, varying both ATP and inhibitor concentrations. Preliminary experiments with palinurin suggest that it might act as a non-ATP competitive inhibitor (data not shown). Different Ircinia dendroides extracts were tested at concentrations ranging from 5 to 50 μg mL −1 on a cell-based phosphorylation assay. In this assay Ircinia dendroides extracts inhibit Tau phosphorylation at concentractions over 50 μg mL −1. When assayed on the Tau phosphorylation cellular assay, both palinurin and tricantin showed signiﬁcant inhibition at a concentration of 200 μM.

16.6

GENISTEINE AND ISOFLAVONES

Estrogen plays a role in the central nervous system through binding estrogen receptors located in the brain and exerting neuroprotective and neurotrophic properties [58]. Epidemiological studies suggest that postmenopausal women using estrogen replacement theraphy (ERT) have a decreased risk of developing dementia. Although ERT alleviates the symptoms associated with menopause and has a positive effect on bones, ERT in postmenopausal women has been linked to a higher incidence of cervical and breast cancer [59]. Consequently the selective estrogen receptor modulators (SERMs) compounds that exert tissue-speciﬁc estrogenic effects may provide the beneﬁts of ERT without the risks. Groups of natural SERMs are the soy-derived phytoestrogens, which are structurally similar to estrogen and may serve as an alternative to ERT.

326

MARINE COMPOUNDS AS A NEW SOURCE FOR GSK-3 INHIBITORS HO

O

OH O

HO

OH

124

Figure 16.5

O

O 125

OH

Genisteine and daidzeine chemical structures.

Soy beans contain a large amount of isoﬂavones, including genistein (4′,5′,7trihydroisoﬂavone), daidzein (4′,7-dihydroisoﬂavone), glycitein (6-methoxydaidzein), and their glycosides. Experimental evidence suggests that soy isoﬂavones possess many therapeutic properties including estrogenic, antioxidant, hypocholesterolemic, as well as the inhibition of cell proliferation and DNA synthesis. Moreover genistein is a known tyrosine kinase inhibitor [60] and possesses pleiotropic actions, some of which may involve transforming growth factor β [61]. Although the evidence for estrogen’s effect on cognition in women with AD is controversial, interest on phytoestrogens has increased due to its ability to selectively activate estrogen receptors. Recently a C. elegans model that expresses human beta amyloid (Aβ) was used to evaluate the pharmacological effect of the soya-derived isoﬂavones genistein, daidzein, and glycitein on Aβ-initiated toxicity and oxidative stress [62]. Moreover a clinical trial is being carried out to evaluate the potential effects of soy isoﬂavone supplements on cognitive function for men and women with AD [63]. Very recently, in our searching for GSK-3 inhibitors from marine organism and microorganisms source, we have discovered that genistein 124 and daidzein 125 (Fig. 16.5) are metabolites of a marine microorganism Micromonospore sp. and that both compounds exert inhibition of GSK-3 [64]. The dose–response curve study for genisteine showed an IC50 of 45 μM, while experiments performed with daidzeine gave 55% GSK-3 inhibition at 50 μM. This activity increases the value of isoﬂavones as therapeutic for AD and discloses a new chemical family as GSK-3 inhibitors.

16.7

CONCLUDING REMARKS

It is well established that natural products play an important role in the discovery and development of new drugs. More recently the importance of the potential of natural marine products as sources of new drugs covering a very wide range of pharmacological effects has increased. In fact the interest in marine compounds for the treatment of neurological disorders appears to be an emerging ﬁeld [11]. The prominent role that marine invertebrates have played in the generation of novel GSK-3 inhibitors, including hymenialdisine, meridianines, indirubines, manzamines, furanosesquiterpenes, and isoﬂa-

REFERENCES

327

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INDEX

Aβ-AChE complexes, Wnt pathway functionality and, 32. See also Amyloid β (Aβ) Aβ neurotoxicity, 30–31 protection from, 35–37 Aβ peptide production, 32 Aβ toxicity, M1 muscarinic receptor activation as protection from, 33 AChE inhibitors, role in GSK-3β regulation, 33–37. See also Cholinesterase inhibitors (ChEI) Activation loop phosphorylation site, 65 AD brain. See also Alzheimer’s disease (AD) increased levels of GSK-3β in, 108 neuropathological abnormalities in, 107 Tau in, 84 AD cytosolic abnormally hyperphosphorylated Tau (AD P-tau), 84 Adenomatous polyposis coli (APC), 10 phosphorylation of, 14 AD-hyperphosphorylated Tau, 113 Adipogenic transcription factors, Wnt inhibition of, 164

ADME studies, 272 AD pathophysiology, role of PPARγ in, 37 Adult stem cells, 156 A-kinase, 86 Tau prephosphorylation by, 89, 93–99 A-kinase anchoring proteins (AKAPs), 12 Alonso, Diana, 307 Alonso, Mercedes, 257 α-helix structure, in PHF formation, 114 Alsterpaullone complex (PDB 1Q3W), 70 Alzheimer’s disease (AD). See also AD entries drug actions in, 228–229 GSK-3β as a clinical target in, 32 GSK-3 connection with, 15, 107–124, 174–175, 244–266 GSK-3 deregulation in, 56 GSK-3 inhibition as a therapy for, 115–116 GSK-3 regulation and, 108–110 GSK-3 substrates relevant to, 114–115 key features in pathogenesis of, 108

Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, Edited by Ana Martinez, Ana Castro, and Miguel Medine Copyright © 2006 John Wiley & Sons, Inc.

333

334 late stages of, 208 M1 agonists and, 33 manzamine A in the treatment of, 320 neurotoxicity induced by, 228, 230 Tau changes in, 108 therapeutics for, 250 types of, 110 Ames Screening Study, 272 Amides, hymenialdisine-related, 310 5-Amino-1,2,4-thiadiazol-3-one derivatives, 270 3-Amino pyrazoles, as GSK-3 inhibitors, 281–305 3-Amino pyrazole scaffold, 304 2-Aminothiazoles, in CDK-2, 288 AMP-dependent protein kinase (PKA), 8 activation by forskolin, 99 Amphetamine hyperactivity, lithium attenuation of, 141–142 Amyloid β (Aβ). See also Aβ entries; β-amyloid downstream effects of, 175 production of, 230 Amyloid hypothesis, 110 Amyloidosis, 250 Amyloid plaques, 174, 244 Amyloid precursor protein (APP), 35, 107–108, 244. See also APP processing Amyotrophic lateral sclerosis (ALS), 181 Anhedonia, 141 Animal development, role of Wnt signaling and GSK-3 in, 26–27 Animal models. See also Mouse models with modiﬁed GSK-3 expression, 203–219 nonmurine, 214–215 Antidepressants, 127–128 downstream therapeutic targets of, 128 effects on GSK-3, 132–133 effects on GSK-3-mediated signaling, 138 Anti-GSK-3 drugs, development of, 182 Anti-inﬂammatory drugs, 37 role in GSK-3β regulation, 33–37 Anti-inﬂammatory effect, of TDZD-8, 273–274 Antimanic effects, of valproate, 137 Antipsychotics, effects on GSK-3mediated signaling, 138–139

INDEX

Apoptosis extrinsic, 232–233 GSK-3β-facilitated, 32 neuronal, 176 regulation by survival factors, 173–174 TNF-α-induced hepatocyte, 211 TNF-induced, 233 Apoptosis modulation, by TDZD-8, 273 Apoptotic processes, GSK-3 nuclear translocation and, 48 APP processing, 110. See also Amyloid precursor protein (APP) AR-A014418, 181 GSK-3 inhibition by, 247–248 in vivo efﬁcacy of, 249–250 pharmacokinetic properties of, 249 in psychiatry, 250 Tau splicing and, 248–249 Argyrophilic grain disease, 113 5-Aryl pyrazolopyridazines cytochrome P450 and microsomal clearance data for, 298–299 identiﬁcation of, 287–291 5-Aryl pyrazolopyridines, identiﬁcation of, 281–286 Asn186, 68 Asp1S1, 68–69 AT-100 epitope, 245 ATP (adenosine triphosphate) binding pocket regions, 282–284 ATP binding site, 68–69 solvent exposed region of, 72 ATP binding site inhibitors, 70 ATP-competitive inhibitors, 70 small-molecule, 177–178 ATP noncompetitive GSK-3 inhibitors, 258. See also 2,4-Disubstituted thiadiazolidinones (TDZDs) Auto-inhibition, by GSK-3 N-terminus, 66–67 Autophosphorylation theory, 64 Avila, Jesús, 107, 203 Axin binding of, 74–76 GSK-3-mediated phosphorylation of, 10–11 phosphorylation state of, 75 Xenopus, 77 Axin-2, 12 Axin binding site, 75 Axonal elongation, 52 Axonal transport, Tau overexpression and, 214

INDEX

Axon-dendrite polarity, 29 Axon morphogenesis, GSK-3 in, 50 Backbone carbonyl, 69 β-amyloid. See also Amyloid β (Aβ) GSK-3 activation by, 56 production of, 110 β-amyloid-induced neurodegeneration, 108 β-catenin epidermal expression of, 161 lithium and, 229 in neural stem/progenitor cells, 28 neurodegeneration and, 29–30 phosphorylation of, 9–10, 11, 52 primed phosphorylation of, 74 Wnt regulation of, 10 in Xenopus embryonic development, 27 β-catenin stabilization, GSK-3 in, 206 Bhat, Ratan V., 243 Bifunctional compounds, role in GSK-3β regulation, 33–37 Binding assays, 196–197 to different receptors, 266 “Binding pocket,” 111 Bioactivities, of furanosesquiterpenes, 324 Bipolar depressed patients, sleep deprivation of, 140 Bipolar disorder. See also Bipolar mood disorder classes of, 126 lithium salts for, 129 valproate for, 136–137 Bipolar disorder activity, changes in, 141 Bipolar disorder linkage studies, 140 Bipolar mood disorder, lithium treatment in, 115, 233 Bis-indole alkaloids, 310–319 Blood-brain barrier penetrant IMPase inhibitors, 131 Blood-brain barrier permeability, of TDZDs, 271 Blood glucose, orally bioavailable GSK-3 inhibitor and, 302–304 Bone morphogenetic protein (BMP), 164, 165 signaling, 157 Bovine Tau, 85 Brain GSK-3, lithium inhibition of, 136 Bridging water molecule, 69

335 6-Bromoindirubin-3′-oxime (BIO), 28– 29, 164–166 Bromo-substituted indirubins, 318 Budd Haeberlein, Samantha L., 243 C-3 analogues, inhibition of hGSK-3-α for, 285 C-5 analogues, inhibition of hGSK-3-a for, 286 Cadherins, neurodegeneration and, 30 CaM kinase II (CaMKII), prephosphorylation of Tau by, 89–91 CaM kinase II α-promoter, 206 Cancer stem cells, 156 Cancer treatment kinase-directed small-molecule drugs for, 190 TDZD-8 in, 273 Canonical Wnt pathway, 52 Canonical Wnt signaling, 157–158 in stem cell fate regulation, 163 Carboline, GSK-3 inhibition of, 319 Castro, Ana, 257 C/C genotype, 140–141 CD45 + /Sca1 + muscle progenitor cells, 162–163 cdc2/cdk5 inhibitor, effects of, 97 CDK-2 protein, 66, 69, 263–264. See also Cyclin-dependent kinases (CDKs) 2-aminothiazoles in, 288 substrate binding grooves of, 67 CDK-2 inhibition, 286 by 6-heteroaryl pyrazolopyridines, 298 by indazole analogues, 297 by pyrazolopyridine analogues, 294–295 CDK-2 selectivity, determination of, 290–291 CDK5 protein, prephosphorylation of Tau by, 93 Cell-based assays, for GSK-3 inhibition, 197–198 Central nervous system (CNS). See also CNS disorders; Nervous system patterning; Neuro-entries Wnt signaling pathway in, 28, 161 Chen, Shuibing, 155 Chir025, 180 Cholinergic function, Aβ neurotoxicity and, 33–37 Cholinesterase inhibitors (ChEI), 34, 36

336 Chronic diseases, GSK-3 inhibitors for, 17 Chronic lithium treatment, 247 behavioral effects of, 212 Circadian rhythms, deregulation of, 247 CK-1, prephosphorylation of Tau by, 89–93 CK-Iγ isoform, 11 CK-1-mediated phosphorylation, 11 CK-2-catalysed phosphorylation, xi–xii Clorgyline, 138 Clozapine, effect on GSK-3 mediated signaling pathways, 138–139 CMIP analysis, 268, 269 CNS disorders, lithium and, 246–247 Collapsin response mediator protein 2 (CRMP2), 14, 29, 115 Colombres, Marcela, 25 CoMFA methodology, 268–269 Corticobasal degeneration, 113 Crystal structures GSK-3, 61–82 of GSK-3β inhibitor complexes, 70–73 Cyclic AMP levels, intracellular, 179 Cyclin-dependent kinases (CDKs), 70– 72, 143, 198. See also CDK entries efﬁcacy of indirubins toward, 314 Cytochrome P450 data, 300, 301 for 5-aryl pyrazolopyrid[az]ines and 6-[het]aryl pyrazolopyridines, 298–299 Cytoskeletal remodeling, neurite extension and, 46–47 Cytoskeleton, lysophosphatidic acid inﬂuence on, 54–55 Cytotoxicity, rescue of neurons from, 37 Del Monte-Millán, María, 189 Depression, 126–127 medications useful for, 127–128 Developmental signaling pathways, 157 Diabetes. See also Insulin entries GSK-3α and GSK-3β levels in, 211 type 2, 14, 211 Diacylglycerol (DAG), 130 Dickkopf-1 (Dkk-1), 160 Differentiated cells, survival factors for, 173–174 Ding, Sheng, 155 Disability-adjusted life year (DALY) metric, 126–127 Disease disruption of protein kinase function in, 190

INDEX

GSK-3 treatment of, xvii, 224 stem cell approaches to, 156 Disheveled (Dsh) gene, 52 Disheveled (Dsh) protein, 157 2,4-Disubstituted thiadiazolidinones (TDZDs), 258–259 anti-inﬂammatory effect of, 273–274 biological effects of, 264 development as new drugs, 259 drug-like properties of, 270–271 identiﬁcation of, 260–270 inhibitory impact on kinases, 263–264 neuroprotectant properties of, 274–275 second generation of, 269–270 selectivity for GSK-3β, 265 structure-activity relationships of, 267 synthesis of, 260 testing against GSK-3 activity, 261 therapeutic potential of, 270–275 Dithiazolidindione derivatives GSK-3 inhibition of, 263 structure-activity relationships of, 268 Dopamine (DA) action, mediation of, 212 Dopamine neuron differentiation, 167 Dopaminergic (DA) neurons, 26 Drosophila genetic epistasis analysis in, 12 genetic experiments in, 11 Tau overexpression in, 245–246 for transgenic neurodegeneration studies, 214 Wingless (Wnt) signaling pathway in, 27, 73 Drug design, three-dimensional structure-based, 191 Drug discovery, protein kinase assays for, 189–201 Drug-like properties, of TDZDs, 270–271 Drugs, role of natural products in the development of, 326–327 Drug targets, protein kinases as, 189–191 DYRK, activation loop autophosphorylation in, 64 Electroconvulsive seizure (ECS), effects on GSK-3 mediated signaling, 138 Electroconvulsive therapy (ECT), 138 Embryoid bodies (EBs), 156–157 Embryonic stem cells (ESCs), 155–156

INDEX

modulation by Wnt signaling components, 27–28 self-renewal of, 164–166 Endogenous regulatory mechanisms, 222 Endothelial differentiating gene (EDG), 54 Engel, Tobias, 203 Enzyme inhibitors, identiﬁcation of, 226 Enzymes, lithium inhibition of, 130 Epidermal stem cells (EPSCs), Wnt signaling and, 160–161 Estradiol signaling, GSK-3 role in, 53 Estrogen, effects on GSK-3 mediated signaling, 139–140 Estrogen receptor α (ERα), 139–140 Estrogen replacement theraphy (ERT), 325 Extrinsic apoptosis, 232–233 Facci, Laura, 173 Familiar Alzheimer’s disease (FAD), 110–111 Farías, Ginny, 25 d-Fenﬂuramine, 138 Fluorescence-based protein kinase activity assays, 193–195 Fluorescence polarization (FP) kinase assays, 195 Focal ischemia, 176 Forced swim test (FST) antidepressant-like effects in, 142 GSK-3 inﬂuence in, 233 Forskolin, as an A-kinase activator, 93–99 Forster resonance energy transfer (FRET), 195 Frat1, role in Wnt signaling pathway, 76 Frat1 binding site, 75 Frat1 peptide (Frattide), 76–77 FRAT proteins, in GSK-3 control, 50–51 FRB*, 212–213 FRET-peptides, 195 Frizzled (fz) proteins, 52, 53 Frizzled serpentine receptor family, 9, 11 Frontotemporal dementia (FTD), 246 FTDP-17, 205 mutations of, 112–113, 207–208 FTDP-17 Tau, 111 Fuertes, Ana, 189 Furanosesquiterpenoids, 324–325

337 γ-catenin, 30 γ-phosphate transfer, 68–69 γ-secretase activity, 244 Garrido, Juan J., 45 Genetic evidence, for GSK-3 role in mood disorders, 140–141 Genistein, 325–326 Glucose metabolism, GSK-3 and, 13–14. See also Insulin Glutamate-induced excitotoxicity, 274 Glutamatergic neurotransmission, 179–180 Glycine insertion, 72 Glycine-rich loop, 69, 72 Glycogen phosphorylase, ix Glycogen synthase (GS), ix, 66 GSK-3 phosphorylation of, 4–5 phosphorylation of, 85 Glycogen synthase activity, 13 GSK-3 inhibitor effect on, 197 Glycogen synthase kinase 2 (GSK-2), x Glycogen synthase kinase 3 (GSK-3). See also GSK-3 entries; hGSK-3-α in Alzheimer’s disease. 15, 107–124, 174–175, 244–246 in the brain, 131 in cellular processes, 204 crystal structures of, 8–9, 61–82 development of inhibitors of, 143 as a direct target of lithium, 142, 232 discovery of, ix–x distribution in neurons, 47–49 effects of mood stabilizers and antidepressants on, 132–133 functions and future of, xii, 4 genes encoding, 3 glucose metabolism and, 13–14 history of, 4–15 implications in diseases, 204 insulin and, x–xi, 14, 246 interest in, xvii, 83–84 involvement in the Wnt pathway, 9–12 isoforms of, 47 lithium inhibition of, 131–136 in microtubule coordination, 15 in neurodegeneration/ neuroprotection, 173–187 neuronal functions of, 14–15 noninterchangeable isoforms of, 182 phosphorylation of substrates, 224–225 pro-apoptotic effects of, 231–232 protein kinase activity of, 4 role in animal development, 26–27

338 role in neuronal development, 47–49 role in neuronal morphogenesis, 49–50 role in neuronal polarity, 50 serine phosphorylation of, 51, 52 stem cells and, 155–171 structure and function of, 8–9 substrate speciﬁcity of, xi–xii, 199 support for role in mood disorders, 140–142 as a target for indirubins, 314 as a target for mood disorder treatments, 125–154 Tau phosphorylation by, 84–85, 85–87 Glycogen synthase kinase 3β (GSK-3β), 25–43. See also GSK-3β entries chemical inhibitors of, 27 as a clinical target in Alzheimer’s disease, 32 inactivation of, 26 neuronal polarity and, 29 Glycogen synthesis, lithium and, 225 GOLD docking studies, 292 Gómez-Sintes, Raquel, 203 Gonadal steroids, 139 Gould, Todd D., 125 G proteins, heterotrimeric, 54–55 Grundke-Iqbal, Inge, 83 GSK-3 activation, 55–56. See also Glycogen synthase kinase 3 (GSK-3) putative physiopathological implications of, 56 GSK-3 activity control of, 50–51 detection in cultured cells, 197 in vitro, 261 long-term genetic suppression of, 212–213 in neurons, 51–55 GSK-3α gene, 6–7 GSK-3α protein N- and C-termini of, 61 in neuronal compartments, 48 phosphorylation sites of, 63 GSK-3β activation, in neurodegeneration, 29–32 GSK-3β ATP binding site inhibitors, 70 structures of, 71 GSK-3β gene, 6–7 deregulation of, 204 GSK-3β heterozygous knockout mice, 211–212 GSK-3β inactivation, 33

INDEX

muscle glycogen synthase activation and, 213–214 GSK-3β inhibition, selectivity of pyridazine 28 and 32 for, 290 GSK-3β inhibitor complexes, crystal structures of, 70–73 GSK-3β inhibitors, chemical structure of, 165 GSK-3β-K85R mutant, 64 GSK-3β kinase domain, ﬂexibility of, 72–73 GSK-3β protein axonal polarity and, 50 crystal structures of, 61–62 deregulation of activity of, 108 hippocampal overexpression of, 206 inactivation of, 54 inhibition by palinurin, 325 lithium inhibition of, 226 N- and C-termini of, 62 in neuronal compartments, 48 overexpression of, 175 phosphorylation sites of, 62–64 selectivity of pyrazolo[3,4-b]pyridines for, 287 selectivity of TDZDs for, 265 substrate binding grooves of, 67 as a therapeutic target, 77 GSK-3β regulation, role of bifunctional compounds, AChE inhibitors, and anti-inﬂammatory drugs in, 33–37 GSK-3β speciﬁc inhibitors, 64 GSK-3β transgenes, 204–205 GSK-3 catalyzed Tau phosphorylation, by non-proline-dependent protein kinases, 85–87 GSK-3 inhibition, x–xi, 15, 16–17 functional cell-based assays for, 197–198 lithium, 131–136, 176–177 by manzamine analogues, 321–323 rodent studies of, 135 therapeutic applications of, 233–235, 243–255 as a therapy for Alzheimer’s disease, 115–116 GSK-3 inhibitors. See also Lithium entries; Orally bioavailable GSK-3 inhibitor 3-amino pyrazoles as, 281–305 ATP pocket targets for, 70 downstream effects of, 198 marine compounds as a source for, 307–331

INDEX

search for, 258 selective and ATP noncompetitive, 257–280 selectivity of, 198–199 selectivity proﬁ ling of, 290 small-molecule, 177–181, 235, 250, 299 stem cells and, 164–168 as therapeutic agents, 191 therapeutic potential of, 250–251 use of, 167–168 GSK-3 isoenzymes, 109 GSK-3 isoforms, 243–244. See also GSK-3α entries; GSK-3β entries GSK-3 kinase activity, pathways that modulate, 49 GSK-3 knockout mice, 13 GSK-3 mediated signaling effects of antidepressants on, 138 effects of antipsychotics on, 138–139 effects of electroconvulsive seizures on, 138 effects of estrogen on, 139–140 effects of thymoleptic medications on, 136–140 effects of valproate on, 136–137 GSK-3 modulation, cellular responses to, 177 GSK-3 molecules, mammalian, 6–7 GSK-3 N-terminus, auto-inhibition by, 66–67 GSK-3 overexpression, in mouse models, 204–211 GSK-3 polymorphisms, 140 GSK-3 protein, 108 GSK-3 regulation, 7–8, 224–225 Alzheimer’s disease and, 108–110 GSK-3 residues, phosphorylation of, 51 GSK-3/shaggy, role in neuronal cell biology, 45–59. See also Shaggy/ zeste-white3 (sgg/zw3) gene GSK-3 substrate phosphorylation, 4, 64–66 GSK-3 substrates, 5–6 relevance to Alzheimer’s disease, 114–115 GSK-3 tyrosine phosphorylation, regulation of, 51 “Guided docking” approach, 197 Hair growth, Wnt signaling in, 160–161 Haloperidol, 139 Hematopoietic stem cells (HSCs), 156. See also HSC niches Wnt signaling and, 158–159

339 Heritability estimates, for bipolar disorder and depression, 127 Hernández, Félix, 107, 203 hESCs (human embryonic stem cells), maintenance of, 166. See also Human embryonic stem cells 6-Heteroaryl pyrazolopyridines cytochrome P450 and microsomal clearance data for, 298–299 inhibition of hGSK-3α and CDK-2 by, 298 structure-based design of, 291–298 Heterocycles, inhibition of hGSK-3-α by, 285 Heterocyclic compounds, TDZDrelated, 260 hGSK-3-α. See also Glycogen synthase kinase 3 (GSK-3) inhibition by 6-aryl-pyrazolopyridine analogues, 292 inhibition by 6-aryl-pyrazolopyridines containing side chains, 294 inhibition by 6-heteroaryl pyrazolopyridines, 298 inhibition by C-5 substituted 6-arylpyrazolopyridine analogues, 293–294 inhibition by indazole analogues, 297 inhibition by pyrazolopyridine analogues, 295 inhibition of, 284–287 Hh signaling, 157 High-throughput screening (HTS), 191, 194 Hinge region, 69 Hippocampal neurons, pre-treatment of, 180 HSC niches, 159 hTau mice, 250 Human embryonic stem cells, Wnt proteins and, 27–28. See also hESCs (human embryonic stem cells) Human immunodeﬁciency virus type 1 (HIV-1), 176 Huntington’s disease, 181 Hydantoine derivatives/hydantoines GSK-3 inhibition of, 263 structure-activity relationships of, 268 Hydrazones, hymenialdisine-related, 310 Hymenialdisine 1 (HD), 308–310 analogues of, 311–312 hydrazones and amides related to, 310

340 structural analogues of, 310 total synthesis of, 309–310 Hyperphosphorylated Tau protein, 91, 113, 206 IBU-PO (ibuproﬁ n-pyrisostigmine), 34, 35 IGF-1 mimetic activity, of TDZD-8, 272 IKKβ-dependent phosphorylation, 274 Imipramine, 138 IMPase inhibitors, 131 Indazole analogues, inhibition of hGSK-3α and CDK-2 by, 297 Indirubin complexes, 70–72 Indirubins, 29, 313–319 natural, 318 selectivity against kinases, 318 Indole alkaloids, 310–319 Inestrosa, Nibaldo C., 25 Inhibitory serine-phosphorylation, lithium promotion of, 227–228 Inositol 1,4,5-trisphosphate (IP3), 130 Inositol monophosphatase, 225 Inositol monophosphate phosphatase (IMPase), 130 Inositol polyphosphate 1-phosphatase (IPPase), 130 Insulin, GSK-3 and, x–xi. See also Glucose metabolism Insulin/IGF1 pathway, GSK-3 role in, 52 Insulin-like growth factor 1 (IGF1), 131, 135 Insulin receptor substrate 1 (IRS-1), 14 protein levels in, 211 Insulin resistance, 14 GSK-3 and, 246 treatment with a PPARγ agonist, 37 Insulin treatment, GSK-3 inhibition from, 16–17 Intestinal stem cells, Wnt signaling and, 159–160 In vitro protein kinase activity assays, 191–197 In vitro tyrosine autophosphorylation, 64 Iqbal, Khalid, 83 Ircinal A, GSK-3 inhibition of, 319 Ircinia dendroides extracts, 325 Ischemia, focal, 176 Isoenzymes, GSK-3, 109 Isoﬂavones, 325–326 Isomeridianins, 313

INDEX

JNPL3 mice, 249 Jope, Richard S., 223 K85R point mutation, 212 Kinase inhibitors, selective, 70–72 Kinase–kinase interactions, in Tau phosphorylation, 93–99 Kinases advances in knowledge of, 77 manzamine A inhibition proﬁ le on, 324 selectivity of indirubins against, 318 Kinase scaffolds, identifying, 304 Kinase selectivity panel, 198 Kinase selectivity studies, 263–264 Kinase-targeted drugs, 190–191 Kinesin, 230 Kinome, 190 Knockout mice, GSK-3, 13 L803-mts, 143 Label-retaining cells (LRCs), 160 Lamotrigine, 137 Lennox-Gastaut syndrome, 137 LIF/interleukin 6 (IL6), 164 Lindquist, Johanna M., 243 Lipinski rule of ﬁve, 271 Lithium (Li). See also Chronic lithium treatment anti-apoptotic actions of, 231 behavioral effects in rodent models, 141 for bipolar disorder, 127 CNS disorders and, 246–247 dual inhibitory regulation of GSK-3, 234–235 GSK-3 inhibition throughout the cell, 224–225 as a GSK-3 inhibitor, 115, 131–136, 176–177, 225–227, 240 levels of, 227 microtubule modulation by, 229–230 molecular effects of, 129–131 as a neuroprotective factor, 32 promotion of inhibitory serinephosphorylation, 227–228 studies of, 234 therapeutic efﬁcacy of, 234 therapeutic window for, 129 toxicity of, 233 various targets of, 228–231 Lithium-activated phosphatidylinositol 3-kinase (PI3K), 228

341

INDEX

Lithium chloride (LiCl), effect on GSK-3 and PKA activities, 97 Lithium-dependent behavior, 142 Lithium salts, for bipolar disorder, 129 lpa1 (EDG2) gene, 54, 55 Lucas, José J., 203 Luminescence-based protein kinase assays, 196 Lys85, 68 Lys183, 69 Lysophosphatidic acid (LPA), neuron morphology regulation by, 54–55 M1 muscarinic receptor activation, as protection from Aβ toxicity, 33 mAb Tau-1, binding of, 87 Magnesium (Mg), GSK-3 inhibition and, 134, 226 Maleimide-related compounds, GSK-3 inhibition of, 262 Maleimides, structure-activity relationships of, 267 Mammalian GSK-3 molecules, 6–7 Mania lithium for, 129 medications useful for, 127–128 Manji, Husseini K., 125 Manzamine A, 319 inhibition proﬁ le of, 324 in the treatment of Alzheimer’s disease, 320 Manzamine analogues, GSK-3 inhibition by, 321–323 Manzamines, 319–324 MAPK (mitogen-activated protein kinase), 94 Mapping studies, CoMFA methodology and, 268–269 Marine compounds, as GSK-3 inhibitor sources, 307–331 Martín-Aparicio, Ester, 189 Martínez, Ana, 257, 307 Medina, Miguel, 189 Meridianins, 312–313 mESCs (mouse embryonic stem cells), maintenance of, 166 Mesenchymal progenitors, Wnt signaling and, 163–164 N-Methyl-d-aspartic acid (NMDA), 179–180 Mice, chronic treatment with haloperidol, 139 Microsomal clearance data, 300, 301

for 5-aryl pyrazolopyrid[az]ines and 6-[het]aryl pyrazolopyridines, 298–299 Microtubule-associated protein 1b (MAP1b), 14 Microtubule-associated proteins (MAPs), 46–47, 229 phosphorylation level of, 55 Microtubule binding domain (MBD), 111 Microtubule binding proteins, 115 Microtubule-interacting proteins (MIPs), 46 Microtubules (MTs), 46 lithium modulation of, 229–230 NGF regulation of, 54 stimulation of assembly and maintenance of, 93 Midbrain-hindbrain boundary (MHB) formation, Wnt signaling in, 167 Mitochondrial GSK-3, 225 Mitsunobu reaction modiﬁcation, 260 Modiﬁed GSK-3 expression, animal models with, 203–219 Modiﬁed GSK-3 forms, mouse models with, 213–214 Mood disorders, factors in, 127 Mood disorder treatments GSK-3 as a target for, 125–154 limitations of current, 126–128 outcomes of, 143 Mood stabilizers, 247 downstream therapeutic targets of, 128 effects on GSK-3, 132–133 Mouse mammary tumor virus, 9 Mouse models. See also Rodent entries GSK-3 overexpression in, 204–211 with modiﬁed GSK-3 forms, 213–214 with reduced GSK-3 activity, 211–213 “Multisite” phosphorylation, ix Mutations, in β-catenin, 10 Myc proteins, 166 Myeloma cell growth, 272–273 Myogenic progenitor cells (MPCs), Wnt signaling and, 162–163 Myoinositol, 130 Navarro, Luisa, 189 Nervous system patterning, Wnt signaling in, 166–167. See also Central nervous system (CNS) Neural crest development, Wnt signaling in, 28

342 Neural crest stem cells (NCSCs), Wnt signaling and, 162 Neural differentiation, Wnt signaling and, 161–162 Neural progenitor cell differentiation, 166–168 Neural stem cells Wnt3a and, 28 Wnt signaling and, 161–162 Neurite extension, cytoskeletal remodeling and, 46–47 Neurite growth, control of, 46 Neuritic dystrophy, 245 Neuritogenesis, 46 Neurodegeneration amyloid-induced, 248 GSK-3 and, 174–176 GSK-3β activation in, 29–32 GSK-3 substrates related to, 114–115 Neurodegenerative diseases/disorders characteristics of, 174 GSK-3 activation in, 175–176 Neuroﬁbrillary tangles (NFTs), 107– 108, 244. See also NFT pathology Neurological processes, GSK-3 role in, 143 Neuronal apoptosis, 176. See also Neuronal cell death Neuronal cell biology, role of GSK-3/ shaggy in, 45–59 Neuronal cell death, glutamate-induced, 179–180. See also Neuronal apoptosis; Neuronal death Neuronal compartments, GSK-3α and β in, 48 Neuronal death, TDZD protection against, 264 Neuronal development, GSK-3 in, 47–49 Neuronal functions, 14–15 Neuronal morphogenesis, role of GSK-3 in, 49–50 Neuronal polarity GSK-3β and, 29 GSK-3 role in determining and maintaining, 50 Neuron morphology, lysophosphatidic acid regulation of, 54–55 Neurons degeneration of, 244 GSK-3 distribution in, 47–49 pathways controlling GSK-3 activity in, 51–55 Neuroprotection GSK-3 inhibitors and, 176–181

INDEX

by lithium, 115, 231 by PPARγ activation against Aβ toxicity, 37 by TDZDs, 274–275 Neurotoxicity lithium and, 177 6-OHDA, 274 NF-κB activity, inhibition of, 309. See also Nuclear factor-κB entries NF-κB pathway, GSK-3 and, 274 NFT pathology, 246, 249. See also Neuroﬁbrillary tangles (NFTs) NGF signaling, GSK-3 role in, 53–54 N-methyl-d-aspartic acid (NMDA), 179–180 Noncanonical Wnt signaling, 158 hematopoietic stem cells and, 159 Non-insulin-dependent diabetes mellitus (NIDDM), 246 Nonmurine animal models, 214–215 Non-proline-dependent protein kinases, 85–87 Nonsteroidal anti-inﬂammatory drugs (NSAIDs), role in GSK-3β regulation, 35 Notch signaling, 157 N-terminal phosphorylation, 9, 12 Nuclear factor-κB (NF-κB) activation, 211. See also NF-κB entries Nuclear factor-κB (RANK) receptor, 163 6-OHDA, neurotoxicity of, 274 Orally bioavailable GSK-3 inhibitor, efﬁcacy data for, 299–304 Osteoblasts, Wnt signaling and, 159 Osteoprotegerin (OPG), 163 Paired helical ﬁ laments (PHFs), 245 Palinurin, 324–325 Pancreatic tumor cells, inhibition of GSK-3 in, 13 Parkinson’s disease, 176 Pathological conditions, GSK-3 dysregulation in, 15 PD 98059, effects of, 98 PEGION screens, 70 Peptides, Wnt signaling pathway, 73–77 Pérez-Puerto, José, 189 Peroxisome proliferator-activated receptor (PPARs), 37. See also PPARγ entries Pharmacokinetic and pharcodynamic (PK/PD) properties, of TDZDs, 271

INDEX

Pharmacophore model, 282–283 Phe67 residue, 69 PHF-1 site, phosphorylation of Tau at, 94 PHF particle, molecular structure of Tau protein in, 114 Phosphatidylinositol (PI) 3′ kinase (PI3K), xi, 7. See also PI3K entries lithium-activated, 228 signal transduction pathway, 174 Phosphatome, 190 3-Phosphoinositide-dependent protein kinase 1 (PDK1), xi, 7 Phosphoinositol signaling, downstream targets of, 130 Phosphomonoesterases, 130 lithium inhibition of, 247 Phosphorylase kinase, ix Phosphorylated Ser9, 66–67. See also Ser9 phosphorylation Phosphorylated Tau peptides, ﬁbrillar aggregates of, 114 Phosphorylation, as a major physiological mechanism, 224 Phosphorylation sites, of GSK-3β, 62–64 Phospho-Tau, 111, 112. See also Phosphorylated Tau peptides Phytoestrogens, 326 PI3K/Akt pathway. See also Phosphatidylinositol (PI) 3′ kinase (PI3K) activation of, 53–54 activity of, 52 PI3K-inhibiting GSK-3 activity, 110 PI3K inhibitor, 198 Pick’s disease, 113 PIP2 signaling, 130 PKC isoenzymes, exposure to Aβ, 30–31 Platelet-derived growth factor (PDGF) β-chain promoter, 205–206 PNU112455A, co-injection of forskolin with, 95, 97 PPARγ activation, neuroprotection by, 37. See also Peroxisome proliferator-activated receptor (PPARs) PPARγ nuclear factor, 274–275 PPPSP motifs, 11, 12 Preselinin proteins, 29, 110 Priming kinase, xii, 66 Priming phosphorylation, xii, 4–5, 8–9, 64–66, 77, 225

343 Prion diseases, 176 Pro-apoptotic stimuli, protection from, 174 Progressive supranuclear palsy, 113 Proline-directed protein kinases (PDPKs), 84 Protein Data Bank, 62, 70 Protein function, three-dimensional atomic structures of, 62 Protein kinase A (PKA), ix, x Protein kinase activity assays. See also Protein kinase assays ﬂuorescence-based, 193–195 in vitro, 191–197 Protein kinase assays binding assays, 196–197 for drug discovery, 189–201 luminiscence-based, 196 radioactivity-based, 192–193 Protein kinase B (PKB), xi, 7–8, 84 Protein kinase C (PKC), muscarinicreceptor-induced activation of, 33 Protein kinase C isozymes, selective reductions of, 130–131 Protein kinase function, disruption of, 190 Protein kinase genes, 190 Protein kinase inhibition, selectivity of, 263–264 Protein kinase inhibitors (PKIs), x selectivity of, 198–199 Protein kinase pharmacophore model, 282–283 Protein kinases as drug targets, 189–191 non-proline-dependent, 85–87 Protein phosphatase 1 (PP1), lithium-reduced activity of, 228 Protein phosphorylation, 85, 189–190 PrP 106–126, 176 PS-1 mutation, 110 Psychiatry, AR-A014418 in, 250 Pyrazolo[3,4-b]pyridazine, co-crystallisation with GSK-3, 289 Pyrazolopyridazines, inhibition of hGSK-3-α by, 284, 289–290 Pyrazolopyridine 92, selectivity proﬁ ling of, 298 Pyrazolopyridine analogues, inhibition of hGSK-3a and CDK-2 by, 284, 295 Pyrazolopyridines, selectivity proﬁ ling of, 296 Pyridazine 1, 283

344 Pyrrole alkaloids, 308–310 Pyruvate dehydrogenase, ix Radioactivity-based protein kinase assays, 192–193 Receptors, binding assays to, 266 Recombinant Wnt3, 165–166 Reduced GSK-3 activity, mouse models with, 211–213 Reelin pathway, GSK-3 role in, 54 Reporter genes, TCF-dependent transcriptional activation of, 198 Resting cells, 4 Rhodanine-related derivatives, GSK-3 inhibition of, 262 Rhodanines, structure-activity relationships of, 267–268 Risperidone, 139 Rodent behavioral models, as support for GSK-3 role in mood disorders, 141–142. See also Mouse entries Rodent studies, of GSK-3 inhibition, 135 Rp-cAMPS, co-injection of forskolin with, 95, 97 S9A mutant form, 205 SB 203580, effects of, 98 SB-216763/415286 GSK-3 inhibitors, 177–179, 180, 181 SC35 splice factor, 249 Scintillation proximity assay (SPA), 192, 194 Second-generation TDZDs, 269–270 “Second messenger,” x Selective estrogen receptor modulator (SERM) compounds, 325 Senile plaques (SP), 107–108 Ser9 phosphorylation, 33, 37. See also Phosphorylated Ser9; Serine phosphorylation Ser9 phosphorylation site, 62 Ser9/21 phosphorylation, inhibition of, 109 Ser214, phosphorylation of Tau at, 94 Ser262/356 kinases, 111 Serine-phosphorylated GSK-3, 224, 234–235 Serine phosphorylation, 51, 53. See also Inhibitory serine-phosphorylation “feedback mechanism” for, 134 of GSK-3, 224, 234–235 Serine/threonine-speciﬁc phosphatase, xi

INDEX

Shaggy/zeste-white3 (sgg/zw3) gene, 7, 112. See also GSK-3/shaggy Signaling cascades, 53 Signaling molecules, pools of, 12 Signaling proteins, GSK-3β-regulated, 175 Signal selectivity, 12–13 Simón, Diana, 45 Site–site interactions, in Tau phosphorylation, 87–93 Site-speciﬁc modulation, of tau phosphorylation, 87–93 Skaper, Stephen D., 173 Small-molecule GSK-3 inhibitors, 177– 181, 235, 250, 299 SMI31 epitope, 88, 89 SMI34 epitope, 91 Soy isoﬂavones, 326 Splice isoform, of GSK-3β, 7 Staurosporine-induced apoptosis, 274 Stem cell biology, advances in, 156 Stem cells. See also Embryonic stem cells (ESCs) classes of, 155–156 differentiation of, 156–157 epidermal, 160–161 GSK-3 and, 155–171 hematopoietic, 158–159 intestinal, 159–160 neural, 161–162 neural crest, 28, 162 Wnt signaling and, 157–164 Structure-activity relationship (SAR), of manzamines, 320 Substrate-binding groove, 72–73 Survival factors, 173–174 SXXXpS canonical phosphorylation motif, 66, 67 Tau-1 binding, decrease in, 88–89 Tau aggregation, 113–114, 230 Tau antibodies, binding pattern of, 90–91 Tau hyperphosphorylation, 30, 55, 112, 113, 206 lithium inhibition of, 177 lithium modulation of, 115 Tau mutations, 112–113, 207–208 Tauopathies, 112–113. See also Tau pathologies GSK-3 and, 174–175 Tau overexpression, in Drosophila, 214 Tau pathologies, GSK-3 inhibitors in, 115. See also Tauopathies

345

INDEX

Tau phosphorylation, 15 in Alzheimer’s disease, 111 AR-A014418 inhibited, 181 by GSK-3, 113–114 kinase–kinase and site–site interactions in, 83–103 lithium and, 230 modulation of GSK-3 catalyzed, 85–87 persistent, 245 site-speciﬁc modulation of, 87–93 TDZDs and, 264 in transgenic mouse lines, 208–211 Tau protein. functions of, 84 hyperphosphorylated, 30, 55, 112, 113, 206 overexpression of, 175 phosphorylation of, 66 prephosphorylated by A-kinase, 93–99 prephosphorylation of, 88–93 Tau protein kinase I, as a PI 3-kinasemediated neuronal cell survival effector, 174 Tau splicing, AR-A014418 and, 248–249 T-cell factor/lymphoid enhancerbinding factor (TCF/LEF), 26 TDZD-related heterocyclic compounds, synthesis of, 260. See also 2,4Disubstituted thiadiazolidinones (TDZDs) Ter Haar, Ernst, 61 Tet/GSK-3β mice, 206, 207 Tet/GSK-3β/VLW mice, 208 Tetracycline-regulated system, 206 Therapeutic agents, GSK-3 inhibitors as, 191 Therapeutic applications, of GSK-3, 233–235 Therapeutic potential of 2,4-disubstituted thiadiazolidindiones, 270–275 of GSK-3 inhibitors, 250–251 Therapeutic strategy, GSK-3 inhibition as, 243–255 Thiadiazolidinones (TDZD), 77, 115, 196. See also 2,4-Disubstituted thiadiazolidinones (TDZDs) glycogen synthase kinase 3β inhibition of, 259 Thiazolidinedione drugs, 37 Thiazolidines, structure-activity relationships of, 267–268

Three-dimensional atomic protein structures, 62 Three-point binding modes, 284 Three-point hydrogen bond, 288 thy1 gene, 205 Thymoleptic medications, effects on GSK-3 mediated signaling, 136–140 Time-resolved FRET (TR-FRET), 195 Tissue-selective GSK-3 inhibition, 235 TNFα sensitivity, 13. See also Tumor necrosis factor entries Transcription factors, as GSK-3 targets, 232 Transgenic ﬂ ies, 112–113 Transgenic mouse lines, with modiﬁed GSK-3 expression, 204–210 Transgenic Tau, suppression of, 116 Transit amplifying (TA) cells, 160 Triazolidinedione derivatives, 260 GSK-3 inhibition of, 263 Triazolidinediones, structure-activity relationships of, 268 Tricantin, 324–325 Trinitrobenzene sulphonic acid (TNBS), 273 T/T genotype, 140 Tubulin-binding proteins, 46–47 Tumor necrosis factor-α (TNF-α)induced hepatocyte apoptosis, 211. See also TNFα sensitivity Tumor necrosis factor (TNF)-induced apoptosis, 233 Tumors, APC, β-catenin, and axin in, 10 TUNEL, 205, 206 12E8 epitope, 111 TWS119, 167 Type 2 diabetes mellitus (T2DM), 14 GSK-3α and GSK-3β levels in, 211 Tyr216/276, activation of, 109 Tyr216 phosphorylation, 176 Tyr216 phosphorylation site, 62–64 Tyrosine autophosphorylation, 64 Tyrosine kinase PYK2, 64 Tyrosine phosphorylation, 7, 8, 51 of GSK-3, 224 Unipolar depression, 126 Valproate, effect on GSK-3 mediated signaling, 136–137 Valproate derivatives, 137 Varea, Olga, 45

346 Virtual screening, 196–197 VLW mice, 208 Wandosell, Francisco, 45 Wingless (Wnt), signaling pathway in GSK-3 inhibition, 109–110. See also Wnt entries Witherington, Jason, 281 Wnt1 gene, 9, 26 Wnt3, recombinant, 165–166 Wnt3a neuroprotective effect, 30 Wnt agonists, 166 Wnt antagonists, 166 Wnt/β-catenin pathway, in intestinal stem cell maintenance, 160 Wnt/β-catenin signaling, in neural crest stem cells, 162 Wnt/Ca2 + pathway, 158 Wnt ligand/ligation, 12, 26 Wnt pathway, GSK-3 involvement in, 9– 12, 52–53 Wnt pathway functionality, Aβ-AChE complexes and, 32 Wnt/PCP pathway, 158 Wnt proteins, 157 Wnts, limitations of, 26–27 Wnt signaling, 15–16 β-catenin-dependent, 138 canonical and noncanonical, 157–158 epidermal stem cells and, 160–161 hematopoietic stem cells and, 158–159

INDEX

intestinal stem cells and, 159–160 mesenchymal progenitors and, 163–164 myogenic progenitor cells (MPCs) and, 162–163 neural stem cells and, 161–162 role in AD pathology, 31 role in animal development, 26–27 stem cells and, 157–164 as a target for AD treatment, 32 Wnt signaling components, embryonic stem cell modulation by, 27–29 Wnt signaling pathway(s), 26 drug modulation of, 27 major components of, 73–74 neurodegenerative disorders and, 29 in regulating cell fate, 164 valproate activation of, 137 Wnt signaling pathway peptides, structures with, 73–77 Wnt target genes, 26 Woodgett, James R., 3 Xenopus embryonic development, 27 Xenopus retina, Sox2 expression in, 161 Zebraﬁsh model, synapse formation in, 214–215 Zinc (Zn) behavioral studies of, 142 inhibition of GSK-3, 134