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 mela-nogaster, 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...

Structure Activity Relationships

TDZD The interesting enzymatic profile 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-3P 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...

Gsk3 A Key Player In Alzheimers Disease

Centro de Biolog a Molecular Severo Ochoa CSIC UAM, Madrid Contents 6.2 GSK-3 Regulation and Alzheimer's Disease 6.3 Alzheimer's Disease, Genetics, GSK-3, and Tau Phosphorylation 6.4 Tau Phosphorylation by GSK-3 6.5 GSK-3 and Other Tauopathies 6.6 Tau Phosphorylation by GSK-3 and Tau Aggregation 6.7 Other GSK-3 Substrates Relevant to Alzheimer's Disease 6.8 GSK-3 Inhibition as a Therapy for Alzheimer's Disease

References

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., Martinez 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...

Pathways Controlling Gsk3 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 relative importance of GSK-3 remains to be...

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, Ap, 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...

Ecs

Bipolar disorder Primary treatment for bipolar disorder Treatment for depression and bipolar disorder Model of Electroconvulsive Therapy (ECT), the most efficacious therapy for severe refractory depression also a successful treatment for refractory mania Direct inhibitor of GSK-3 by competition for magnesium Some studies suggest direct inhibition many studies suggest indirect inhibition Attenuates staurosporin-stimulated-caspase-3 activity in neuroblastoma cells overexpressing GSK-3 Fluoxetine,...

R170

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...

R

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...

Info

In an effort to identify new kinase inhibitors with increased potency and selectivity, natural indirubins produced by the Mediterranean molusk Hexa-plex trunculus were investigated. Several bromo-substituted indirubins (66-76) were identified, the most acitive being a GSK-3P 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...

M

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 M forskolin alone, or forskolin combined with 200 M PNU 112455A. The activities of GSK-3 (A), PKA (B), cdc2 (C), and cdk5 (D) were measured by using specific peptide substrates. The phos-phorylation levels of Tau at various sites were determined by western blots using...

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 histo-logical 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 endopheno-types 124 ), predict the response of future medications based on the effects of existing drugs, or modulate...

Therapeutic Applications Lithium Or Other Inhibitors Of Gsk3

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...

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-3P and CDK5 p25 (Table 16.2) 23 . In addition, derivatives obtained by substitution of bromopyrrole ring (compounds 31-34), show reduced potency against GSK-3P. However, deeper analysis could prove that favorable selectivity toward GSK-3P as against CDK5 p25 can be achieved. For example, hydrazone 28 is an...

Newer Target Of Lithium Direct And Indirect Inhibition Of Glycogen Synthase Kinase

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 first characterized for its role in glycogen synthesis. These findings 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...

Cytochrome P450 And In Vitro Microsomal Clearance Data For 5aryl Pyrazolopyrid [azines And 6[hetaryl Pyrazolopyridines

In order to understand the potential developability liabilities, a number of diverse compounds from the various inhibitor classes were assessed for inhi- 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...

Sitespecific 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 prephos-phorylated 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...

Gsk3 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, apop-tosis, 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...

Other Gsk3 Substrates Relevant To Alzheimers 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 hyperphos-phorylation. Some of these proteins (i.e., P-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...

Specific Gsk3 Inhibitor Ara014418

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...

Glycogen Synthase Kinase 3 Role In Neurodegeneration And Neuroprotection

Neurology and GI Centre of Excellence for Drug Discovery GlaxoSmithKline Research and Development Limited Harlow, Essex, UK 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 9.3.2 Small-Molecule Inhibitors

Lithium Directly And Selectively Inhibits Gsk3

A very large number of biochemical effects of lithium have been reported, but few direct targets are known 13 . The first 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...

AbAche Complexes Induce A Loss Of Function Of The Wnt Pathway

Previously it was shown that 1 to 10 M of Ap fibrils and Ap-AChE complexes induce neuronal cell death in neuronal cell lines and primary cultures of rat hippocampal neurons and chick retina cells. The Ap-AChE complexes neuro-toxicity showed more damage than Ap 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 Ap-AChE complexes. Primary...

Gsk3 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...

Role Of Gsk3shaggy In Neuronal Cell Biology

Diana Sim n, Olga Varea, Juan Jos Garrido, and Francisco Wandosell Centro de Biolog a 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 3.5.6 LPA as Regulator of Neuron...

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...

In vitro GSK3 Activity

Several compounds belonging to the TDZD family or to the previously described chemical related groups were tested against GSK-3P. This activity was determined using recombinant GSK-3P 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...

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. most implicated in this Tau pathology, is the...

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...

Synthesis of 24Disubstituted Thiadiazolidinones

Thiadiazolidinones 1-21 were synthesized following a pathway that is based on the reactivity of N-alkyl-S- 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-oxo-1,2,4-thiadiazolidin-3-thione derivatives 22-24 could be detected as minori-tary...

Tau Becomes A More Favorable Substrate For Gsk3 When Prephosphorylated By Akinase In Vivo In Rat Brain

Forskolin is a well-known specific activator of A-kinase 41,42 . Phosphory-lation of Tau at Ser214 in rat hippocampus increases in a dose-dependent manner after intracerebroventricular (ICV) injection of forskolin 43 . TABLE 5.1 Binding to microtubules of t3L before and after phosphorylation by different kinases TABLE 5.2 Comparison of t sites phosphorylated by A-kinase, GSK-3, or a combination of the two kinases TABLE 5.2 Comparison of t sites phosphorylated by A-kinase, GSK-3, or a...

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...

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-first 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...

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 first 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...

Tdzd

Figure 14.5 Second generation of TDZD. groups were substituted by amino and alkyl groups (Fig. 14.5) 39 . The common final 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-imino-1,2,4-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...

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...

Kinasekinase And Sitesite Interactions In The Phosphorylation Of Tau By Gsk3

Department of Neurochemistry, New York State Institute for Basic Research, in Developmental Disabilities, Staten Island, New York 5.2 Modulation of GSK-3 Catalyzed Phosphorylation of Tau by Non-Proline-Dependent 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

P

Of linearly polarized light are selectively excited. When a fluorescent dye is attached to a small, rapidly rotating molecule, the initially photoselected ori-entational 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...

Abc

Figure 15.3 (A) X-ray of 2-aminothiazoles in CDK-2 (B) model of pyrazolopyrid-azines in GSK-3 (C) overlay of A and B. Figure 15.3 (A) X-ray of 2-aminothiazoles in CDK-2 (B) model of pyrazolopyrid-azines in GSK-3 (C) overlay of A and B. Figure 15.4 X-ray co-crystallisation of a pyrazolo 3,4-b pyridazine with GSK-3. See color plates. Figure 15.4 X-ray co-crystallisation of a pyrazolo 3,4-b pyridazine with GSK-3. See color plates. TABLE 15.7 Selectivity of pyridazine 28 and 32 for GSK-3P...

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 3-position on the pyrrole ring has little effect on the activity of these compounds against GSK-3 p (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...

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...

Introduction To Gsk3

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-3a and GSK-3P, 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,...

Nh

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

Wi Ley1nterscience

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...

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...

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 finding 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 M ATP concentration, and in the presence of increasing meridianin...

Tau Phosphorylation By Gsk3 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-3P is one of the best candidate enzymes for generating the hyperphosphorylated Tau that is characteristic of PHFs. Transgenic animals overexpressing GSK-3P have AD-hyperphosphorylated Tau, although no filaments could be detected 41 . However, when the...

Ara014418

Figure 9.1 Chemical structure of known GSK-3P inhibitors. activity in vitro with IC50s of 78 nM and 74 nM, respectively, and show similar potency toward purified GSK-3P. 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-phosphoinositide-dependent protein kinase-1 65 . SB-216763 and SB-415286 are potent promoters of the survival of cerebellar granule neurons and sensory...

Cytoskeletal Remodeling Associated With Neurite Extension

The nervous system is a highly ordered network of neural connections. These connections are first 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...

Wnt and Epidermal Stem Cells EPSCs

Mammalian epidermis, which is a far more complex structure than the crypt-villus 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 label-retaining cells (LRCs). LRCs are concentrated in the bulge area and express markers such as CD34 and keratin 15 (K15) 25 ....

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...

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...

A

Figure 2.1 The Wnt signaling pathway is a target for AP toxicity. (A) Effect ofAP on Wnt signaling molecules. AP in hippocampal neurons impairs cell survival, increases GSK-3P, decreases cytoplasmic P-catenin and decreases the expression of Wnt target genes. Lithium can attenuate AP neurotoxicity by recovering P-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...

Protein Kinase Assays For Drug Discovery

Miguel Medina, Ana Fuertes, Ester Mart n-Aparicio, Mar a del Monte-Mill n, Mar a-Luisa Navarro, and Mar a Jos P rez-Puerto Neuropharma S.A., Tres Cantos, Madrid 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

Contents

FOREWORD THE ORIGINS OF GSK-3 ix PART I CELLULAR FUNCTIONS OF GSK-3 1 1 Glycogen Synthase Kinase 3 An Introductory Synopsis 3 2 Glycogen Synthase Kinase 3 (GSK-3 ) a Key Signaling Enzyme A Developmental 3 Role of GSK-3 Shaggy in Neuronal Cell Biology 45 4 The Crystal Structures of Glycogen Synthase Kinase 3 61 5 Kinase-Kinase and Site-Site Interactions in the Phosphorylation of Tau by GSK-3 83

Gsk3 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 microtubule-binding 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...

U U

11 14 11 10 11 34 4 0 6 10 9 4 13 0 0 13 12 22 18 1 7 5 11 19 81 90 18 14 10 0 36 045 730 00 0 5 05 14 04 2 05 95 99 22 4 0 0 7 10 0 9 3 9 8 1 0 5 3 2 11 13 4 0 0 10 13 13 99 94 Note Values are I 10 M using 100 M ATP (see 5 for kinases used and assay details). TABLE 15.6 Inhibition of hGSK-3-a by selected analogues

I

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...

SB415286 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 M 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...

Manzamines

Manzamines are complex P-carboline alkaloids isolated from Indo-Pacific sponges and characterized as having an intricate and novel polycyclic system. In 1986, Higa and co-workers first reported manzamine A 101 from the Oki-nawan sponge of the genus Haliclon 47 . To date, 16 species belonging to 8 families of marine sponges, including Acanthostrongylophora, have been confirmed to yield P-carboline manzamine and manzamine-related alkaloids 48 . The occurrence of manzamine alkaloids in a diversity...

Distribution of GSK3 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 profiles of GSK-3 a and GSK-3 p expression are different and in particular, that the p isoform is downregulated after birth. More recent data has shown that GSK-3a and P are expressed in many neuronal compartments and in...

Neuronal Polarity And Gsk30

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-3P plays a...

Z

Fluxation are associated with the onset of mood disorders regulation of estrogen is sometimes therapeutic Amphetamine Can precipitate mania in susceptible individuals used as an lithium responsive rodent model of mania 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 Source Adapted from 155 . See text for discussion and complete references. Increases inhibitory phosphorylation of...

Index

A -AChE complexes, Wnt pathway functionality and, 32. See also Amyloid (A ) A neurotoxicity, 30-31 protection from, 35-37 A peptide production, 32 A toxicity, M1 muscarinic receptor activation as protection from, 33 AChE inhibitors, role in GSK-3 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-3 in, 108 neuropathological abnormalities in, 107 Tau in, 84 AD cytosolic...

Cns Disorders And Gsk3 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...

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...

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 P-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...

Valproate

Valproate (known as valproic acid in the uncarboxylated form), a short-chained 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 reports of valproate utility in acute mania...

The Crystal Structures Of Glycogen Synthase Kinase

Vertex Pharmaceuticals Incorporated, Cambridge, MA 4.1 Crystal Structures of GSK-3 4.2 Phosphorylation Sites of GSK-3 4.3 The Primed-Phosphorylation Mechanism 4.4 Auto-inhibition by GSK-3 N-Terminus 4.6 Crystal Structures of GSK-3 Inhibitor Complexes 4.7 Structures with Wnt Signaling Pathway Peptides

O

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-3a and GSK-3p 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-3P at physiological concentrations in vitro 67 . Similar to lithium, the direct action of zinc appears to be via...

Lithium The Seminal Gsk3 Inhibitor

Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 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

Indole And Bisindole 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. TABLE 16.1 Close analogues of HD, and their biological activity against GSK-3 3, CDK5 p25, and CDKl cyclin B TABLE 16.1 Close analogues of HD, and their biological activity against GSK-3 3, CDK5 p25, and CDKl cyclin B

Putative Physiopathological Implications Of Gsk3 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 (Amyloid 6,28 or PrP 49 . It is for this reason that GSK-3 is emerging as a promising therapeutic target in neurodegenerative processes...

B

Figure 11.1 (A) Tet GSK-3P mice are generated by crossing mice expressing tTA under control of the CamKIIa promoter (tTA) with mice that have incorporated the BitetO construct in their genome (TetO). The double transgenic progeny (Tet GSK-3P) are expected to express GSK-3P constitutively in the brain unless tetracycline or analogues are given orally, thus preventing transactivation by tTA. (B) Hyperphos-phorylation and somatodendritic localization of Tau in Tet GSK-3P mice. PHF-1...

Brief History

GSK-3 was purified as one of five protein kinase activities found to phos-phorylate 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...

Abbreviations

A -Amyloid peptide Abl-K Abelson tyrosine kinase ABP Actin-binding protein ACh Acetyl choline AD P-Tau AD cytosolic abnormally hyperphosphorylated Tau -TRCP -transducin-repeat-containing protein C EBPa CCAAT enhancer binding protein a CaMK II Ca2+ calmodulin dependent kinase II CoMFA Comparative molecular fields analysis CRMP Collapsin response mediator protein DAAM1 Disheveled activator of morphogenesis 1 DMPK Drug metabolism and pharmacokinetics DN Dominant negative Dsh Disheveled DYRK...

Tdzds Selective And Atp Noncompetitive Glycogen Synthase Kinase 3 Inhibitors

Ana Castro, Mercedes Alonso, and Ana Mart nez NeuroPharma Avda. de la Industria, 52, Tres Cantos, Madrid 14.2 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 14.2.4 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...

Gsk3 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-3a and p, 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...

Structurebased Design Of 6[hetaryl 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 group, such as a para or meta phenol moiety at the 6-position of the pyr-azolo 3,4-b pyridine nucleus (Fig....

Gsk3 Inhibition As A Therapy For Alzheimers 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...

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 affinities. 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...

Lithium Salts As Medication

As mentioned, lithium represents a common treatment for bipolar disorder. Lithium salts were first 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 confirmed lithium's efficacy both in the acute...

GSK3b As A Clinical Target In Alzheimers Disease

Neuroprotective targets for AD are still elusive because knowledge of the molecular events that underlie Ap 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-3p activation seems to be a target of Ap-induced apoptosis, different GSK-3p target drugs have been evaluated to avoid the Ap neurotoxicity by cross-talk with the Wnt...

Marine Compounds As A New Source For Glycogen Synthase Kinase 3 Inhibitors

NeuroPharma, Avda. de la Industria 52, Tres Cantos, Madrid Contents 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 16.5 Palinurin and Furanosesquiterpenoids 16.6 Genisteine and Isoflavones

Contributors

De la Industria 52, 28760 Madrid, SPAIN Alonso, Mercedes, NeuroPharma, Avda. De la Industria 52, 28760 Madrid, SPAIN Avila, Jes s, Centro de Biolog a Molecular Severo Ochoa CSIC UAM. Facultad de Ciencias, Universidad Aut noma de Madrid, Cantoblanco, 28049 Madrid, SPAIN Bhat, Ratan, AstraZeneca R& D, S dert lje, Disease Biology, SWEDEN 151 85 Budd Haeberlein, Samantha L., AstraZeneca R& D, S dert lje, Disease Biology, SWEDEN 151 85 Castro, Ana,...

Nfl

Increased tau phosphorylation at AT8 epitope without detectable GSK-3P 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 P-catenin in forebrain spatial learning deficit in Morris Water Maze formation of mature Tau filaments and Tau...

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 phosphory-lation 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. Krebs were awarded the 1992 Nobel Prize...

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+ MPCs is sufficient to induce muscle specification. In addition co-culture with cells ectopically expressing Wnt proteins...

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 Mi muscarinic receptor is expressed in the cerebral cortex and hippocampus, and its major role is in cognitive processing including short-term 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 Ap production 43-35 and also Tau...

Regulation Of Gsk3

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...

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-3P 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...

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...

Atp Binding Site

The ATP binding site is a well defined and fairly deep pocket at the interface of the -terminal and C-terminal domains (Fig. 1A). This binding site is conserved among protein kinases. The Protein Data Bank has two structures of GSK-3P in complex with the nonhydrolyzable ATP analogue AMPPNP (PDB 1PYX and 1J1B) 31,32 . The structures show how the catalytic residues of the y-phosphate transfer area form a large network of interactions with the AMPPNP phosphates (Fig. 4.4A). The binding pocket can...

Table

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 Mi mAChR agonist with functional selectivity preserved both at the level of the Mi mAChR as well as along select signal transduction pathways. c. and d. Rosiglitazone and Troglitazone are thiazolidinedione drugs acting as agonists of PPARy. toxicity, increases the...

I I

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 xM forskolin alone, or forskolin combined with either 100 mM LiCl or 100 M 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....

Lithium Promotes Inhibitory Serinephosphorylation Of Gsk3

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...

Gsk3 As A Therapeutic Target In

6 GSK-3, a Key Player in Alzheimer's Disease 107 7 Glycogen Synthase Kinase 3 A Target for Novel Mood 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 PART III GSK-3 INHIBITORS DEVELOPMENT AND 12 Lithium, the Seminal GSK-3 Inhibitor 223 13 Inhibition of GSK-3 as Therapeutic Strategy in...

Gsk3 In Alzheimers 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 P-amyloid (AP), 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...

How Is Gsk3 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,...

Crystal Structures Of Gsk30 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-3 P 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...