Design of Glycogen Synthase Kinase-3 Inhibitors

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Design of Glycogen Synthase Kinase-3 Inhibitors: An Overview on Recent Advancements Minhajul Arfeen and Prasad V. Bharatam* Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar-160062, Punjab, India Abstract: Glycogen Synthase Kinase-3 (GSK-3) is a constitutively acting multifunctional serine/threonine kinase, a role of which has been marked in several physiological pathways, making it a potential target for the treatment of many diseases, including Type-II diabetes and Alzheimer’s. Design of GSK-3 selective inhibitor was the key challenge which led to the use of rational approaches like structure based methods (molecular docking), and ligand based methods (QSAR, pharmacophore mapping) studies. These methods provide insights into the enzyme–ligand interactions and structure activity relationship of different sets of compounds for the design of promising GSK-3 inhibitors. Molecular dynamic simulation studies have additionally been performed to address key issues like the unique requirement of prime phosphorylation of its substrate at P+4 by GSK-3. An allosteric site has also been reported, where the binding of the peptide leads to the stabilization of the activation loop, resulting in the enhancement of the catalysis of enzymes. These studies are becoming useful in the design of therapeutically active discriminatory GSK-3 inhibitors. In this article, we present a review of recent efforts and future opportunities for the design of selective GSK-3 inhibitors.

Keywords: Glycogen synthase kinase-3, inhibitors design, rationale approaches, selectivity, type-ii diabetes, Alzheimer’s disease, ATP competitive inhibitors, Substrate competitive inhibitors. 1. INTRODUCTION Kinases are enzymes capable of catalyzing the transfer of the phosphate group from high-energy molecules (ATP) to specific substrates. Protein kinases transfers the terminal phosphate group to other proteins resulting in the alteration of their activity [1]. The protein kinases involved in the phosphorylation of serine, threonine and tyrosine residues have been recognized as macromolecular targets for drug discovery [2-6]. GSK-3 transfers the phosphate group from ATP to the hydroxyl side chain of serine and threonine (Ser/Thr specific kinase) of its substrate and results in the inhibition of target proteins [7-9]. GSK-3 attracted much of attention due to its implication in several signaling pathways [10-17] and has been targeted for the development of new therapeutic molecules for the treatment of diseases like Type-II diabetes, Alzheimer’s, chronic inflammatory process, cancer, cardiovascular, and bipolar disorder, etc. [18-33]. GSK-3 is ubiquitously expressed, evolutionarily conserved, constitutively acting serine/threonine specific protein kinase and was originally identified as an enzyme that alters glycogen synthesis via regulation of glycogen synthesis in response to insulin [3438]. GSK-3 is phylogenetically related to other kinases, especially to Cyclin Dependent Kinases (CDKs), Protein Kinase C (PKC), and Mitogen Activated Protein Kinases (MAPKs) [39, 40], it therefore shares high homology in the ATP binding pocket. This has caused the GSK-3 inhibitors to inhibit other protein kinases, even the best among the selective ones have inhibited at least one other kilns in addition to its target [41-43]. Thus the development of bioavailable, potent and selective GSK-3 inhibitors is a big challenge from the drug discovery point of view. During the past two decades, numerous research groups have considered GSK-3 as one of the most potential drug targets for the development of Anti-diabetic and Anti-Alzheimer’s agents, result*Address correspondence to this author at the Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar-160062, Punjab, India; Tel: +91-172-2292018; Fax: +91-172-2214692; Alt: +91-9417503172; E-mail: [email protected] 1381-6128/13 $58.00+.00

ing in the advent of several small molecules [44-55], however no molecule has reached market as yet. Hence, the modulation of existing active molecules to design new molecules, having high selectivity as well as affinity for GSK-3 is an immediate requirement. The selective inhibition of GSK-3 has been a vital concern over the years and different approaches have been taken up to understand underlying factors [56-62]. In this review, we describe recent scientific studies on GSK-3 inhibitors to address three major issues: (i) selectivity requirements of ATP competitive inhibitors, (ii) identification of the new leads, and (iii) optimization of the existing leads. Scope of the article: Considering that GSK-3 is an important and well established target for drug discovery, several reviews have appeared in scientific literature. Among the various published reviews, the emphasis was on biology of GSK-3 [8, 10, 11, 22, 37, 63, 64], GSK-3 in cancer [31, 33, 65, 66], GSK-3 in Alzheimer’s and mood disorder [20, 24], GSK-3 in Type-II diabetes [23], GSK3 inhibitors [15, 19, 21, 67], and clinical status of GSK-3 inhibitors [68]. In one of our previous reviews, structure based methods of GSK-3 inhibitor design were discussed. The focus of the current review is to describe the strategies for GSK-3 inhibitor design. 2. STRUCTURAL DETAILS OF GSK-3 The family of GSK-3 kinase has remained highly conserved throughout the evolution [69, 70] and exists in two isoforms and [71], encoded by two genes, having the molecular weight of 51 kDa and 47 kDa respectively [38]. These two isoforms are almost identical with 98% homology in kinase catalytic domain and overall similarity of 84%. The main difference being the extra glycine stretch present at the N-terminal domain of GSK-3 which is longer by 63 residues [67, 72]. Both isoforms are ubiquitously expressed in mammalian tissues and have similar affinities for their substrates. However, they are not similar in terms of their functions as deficiency of one does not compensate for the other [67, 73-77], which indicates certain independent regulatory roles for and isoforms, but the functional distinction between the two isoforms is yet to be established. Mukai et al. reported another isoform GSK-32 with extra 13 amino acids in the ATP binding domain, which has its own independent regulatory roles in physiological pathways.[78].

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2.1. Crystal Structures of GSK-3 Knowledge of the enzyme substrate interactions obtained from cocrystallized structures has helped in predicting the behavior of newly designed ligands towards protein and therefore, several groups have determined the crystal structure of GSK-3, with the resolution in the range of 1.8-2.9 Å using X-ray diffraction data [79, 80]. GSK-3 exists as a homodimer in the crystallized form with each monomer consisting of a small N-terminal lobe, constituted mostly of -sheets and a large C-terminal lobe formed only of -helices and loops [5, 81]. The catalytic domain (ATP binding pocket) is located between the two lobes. Another important pocket, formed of amino acids Arg96, Arg180 and Lys205 (catalytic triad) is present on the interface of two lobes [82]. As no crystal structure is available for GSK-3, the strategies for design and synthesis of selective inhibitors have been based upon the information obtained from the crystal structures of GSK-3 and homology models of GSK-3. The general features of the GSK-3 understood from the various crystal structures have been discussed in detail in the following sections (Fig. 1).

Arfeen and Bharatam

divided into (i) direct hydrogen bonding interactions and (ii) the water mediated bridged hydrogen bonding interactions. 2.1.1. Polar Interactions The important hydrogen bonding interactions observed in the crystal structures of GSK-3 with its inhibitors are between a carbonyl oxygen of small molecule and the backbone -NH of Val135 while NH center of inhibitor interacts with the carbonyl oxygen of Asp133 (Figs. 2), (4) and (5)). These interactions of inhibitor with hinge residues are important for molecular recognition and have been exhibited by almost every ATP competitive inhibitor of GSK3 reported so far. However, the same pattern of direct hydrogen bonding interaction is not observed in the crystal structures of alsterpaullone with GSK-3 (Fig. 3) in which the carbonyl oxygen of ligand, besides making direct hydrogen bond with the backbone nitrogen of Val135, also interacts with the carbonyl oxygen of Asp133, which is water mediated and makes an additional interaction with hinge residue.

Fig. (2). Staurosporine; PDB code-1Q3D.

Fig. (1). Ribbon representation of GSK-3 PDB code-1Q4L.

So far 34, crystal structures have been reported for GSK-3 with and without ligands. These crystal structures have improved the understanding of interactions made by the enzyme with its substrates and ligand. The list of all the crystal structures with their PDB code, ligands and resolution are tabulated in Table 1. Each monomer of GSK-3 can be divided into two domains. Residues 25-138 at the N-terminal domain comprise mainly of seven antiparallel strands and a small helix (residues 96-102). Residues 139-343 form an -helical domain at the C-terminal, and constitute the activation segment. The ATP binding site that forms the catalytic domain and the substrate binding site, are located at the interface of the -helical and -strand domains and is bordered by the glycine rich loop and hinge. The active site between the two domains is characterized by a channel of approximately 22Å 13Å 15Å dimension, giving a total approximate volume of 4290 Å3 [15]. The interactions made by the GSK-3 and the small molecules at the ATP binding site can majorly be divided into the polar interactions and hydrophobic interactions. Polar interactions can further be

Fig. (3). Alsterpaullone; PDB Code- 1Q3W.

Design of GSK-3 Inhibitors: An Overview on Recent Advancements

Table 1.

Current Pharmaceutical Design, 2013, Vol. 19, No. 00

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Crystallographic Details of GSK-3 Available from PDB

Sr. No.

PDB code

Resolution (Å)

Inhibitor

Binding site

year

1.

1H8F

2.80

None

2.

1GNG

2.60

3.

109U

4.

N.A

2002

Dajani et al. [80]

Fratide Peptide

substrate

2002

Bax et al. [142]

2.40

Axin Peptide

Substrate

2003

Dajan et al. [178]

1Q4L

2.77

Anilinomaleimide

ATP

2003

Bertrand et al. [83]

5.

1Q41

2.10

Indirubin-3’-Monoxime

ATP

2003


6.

1Q3W

2.30

Alsterpaullone

ATP

2003


7.

1Q3D

2.20

Staurosporine

ATP

2003


8.

1PYX

2.40

Amp-Pnp

ATP

2003


9.

1UV5

2.80

6-Bromoindirubin-3’-Monoxime

ATP

2004

Meijer et al. [53]

10.

1Q5K

1.94

Aminothiazole

ATP

2004

Bhat et al. [146]

11.

1R0E

2.25

3-Indolyl-Arylmaleimide

ATP

2004

Allard et al. [179]

12.

2O5K

3.20

Benzimidazole

ATP

2007

Shin et al. [180]

13.

2OW3

2.80

Bisindolylmaleimide

ATP

2008

Zhang et al. [181]

14.

2JLD

1.70

Rytherium Pyridocarbazole

Metal

2008

Atilla et al. [118]

15.

3DU8

2.20

Pyrollopyridinone

ATP

2009

Bossi et al. [182]

16.

3F88

2.60

Methylbenzonitrile+Oxadiazole

ATP

2009

Mol et al. [120]

17.

3F7Z

2.40

Oxadiazole

ATP

2009

Mol et al. [120]

18.

3I4B

2.30

Pyrimidyl Pyrole

ATP

2010

Ter Haaret al. [183]

19.

3LIS

2.90

Pyrazolone

ATP

2010

Haar et al. [82]

20.

3GB2

2.40

Oxadiazole

ATP

2010

Mol et al. [120]

21.

3PUP

2.99

Ruthenium-Octasporine

Metal

2010

Filippakopoulos et al. [184]

22.

3M1S

3.13

Ruthenium Pyridicarbazole

Metal

2010

Atilla et al. [185]

23.

3Q3B

2.70

Pyrimidyl-Pyridones

ATP

2011

Pandit et al. [186]

24.

3ZRM

2.49

Thienopyridinones

ATP

2011

Gentile et al. [187]

25.

3ZRL

2.48

Thienopyridinones

ATP

2011


26.

3ZRK

2.37

Thienopyridinones

ATP

2011


27.

3SD0

2.70

Pyrrolepyridinone

ATP

2011

Mesecar et al. [188]

28.

4DIT

2.60

Imidazopyridine

ATP

2012

Kim et al. [189]

29.

4AFJ

1.98

1,3,4-Oxazolecarboxamide

ATP

2012


30.

4ACH

2.60

Pyrazines

ATP

2012

Xue et al. [127]

31.

4ACG

2.60

Pyrazine

ATP

2012

Xue et al. [122]

32.

4ACD

2.60

Pyrazines

ATP

2012

Xue et al. [122]

33.

4ACC

2.21

Pyrazines

ATP

2012

Xue et al. [122]

34.

3SAY

2.23

Indirubin-3’-Monoxime

ATP

2012

Cheng et al. [191]

2.1.2. Water Mediated Hydrogen Bonding Interactions Water mediated hydrogen bonding interactions sometimes play a very crucial role in stabilizing the crystal complexes by contribut-

Reference

ing in terms of free energy. The crystal structures of GSK-3 enzyme and its inhibitors have also shown some water mediated bridged hydrogen bonds [83]. The water molecules present around


residues Thr138 and Gln185 play major role in stabilizing the complexes of GSK-3. Number of water molecules in the active site depends on the cavity volume left, after the inhibitors occupying their position in the active site. Water molecules present in the active site of GSK-3 have been labeled as wat-1,2,3,4 starting from the vicinity of Thr138 and Gln185 (Fig. 2). Most of the inhibitors have shown interactions with the second water molecule (wat 2) but in the case of maleimide derivatives only first water molecule (wat 1) around Gln185 is present and interacts. This water molecule has been held in its place by Thr138 (Fig. 5). In complexes with oxadiazole and pyrimidyl pyrrole, the third water molecule has also been accommodated in the active site and inhibitors have been found to interact with this third water molecule. In the case of staurosporine complexed with the GSK-3, the second water molecule (wat 2) is a part of the hydrogen bond network that starts with the O atom of the Thr138, passes through four water molecules and terminates at the carbonyl oxygen of Val135 (Fig. 3).

Fig. (4). Indirubin-3-monoxime; PDB Code -1Q41.

Fig. (5). Anilinomaleimide; PDB Code -1Q4L.

Arfeen and Bharatam

Besides these bridged hydrogen bonding interactions, an additional water mediated (wat 5) hydrogen bonding interaction is also present between the carbonyl oxygen of alsterpaullone and Asp133 which is different from the normal hydrogen bonding observed between Asp133 and other inhibitors [83]. 2.1.3. Hydrophobic Interactions The hydrophobic pocket in the ATP binding domain of GSK-3 is formed by the Ile62, Val70, Ala83 on the top and Leu188 at the bottom. The complementary part of the hinge segment consists of Leu132-Asp133-Tyr134-Val135-Pro136 and forms one portion of the pocket while Arg141 forms another part of the pocket by orienting the hydrophobic side chain towards it. The potency of the inhibitors depends on how well the small molecules fit into this hydrophobic region. As observed in the complexes of staurosporine, alsterpaullone, indirubin and 3-anilino-4-arylmaleimide the molecules occupy the surface area of the 891 Å2, 676 Å2, 683 Å2 and 779 Å2 respectively [83]. The occupancy of this additional surface area in the case of staurosporine and 3-anilino-4-arymaleimide may be responsible for their extra potency as compared to other inhibitors. 3. SUBSTRATE SPECIFICITY AND RECOGNITION The two isoforms of GSK-3 share similarity in terms of substrate specificity. Both isoforms require priming phosphate at P+4 position [79], where P is the previously phosphorylated site (serine or threonine), which is either accomplished by priming kinase (first phosphorylation of substrate) or by GSK-3 itself (subsequent phosphorylation). GSK-3 recognizes canonical motif SXXXS(p) where S(p) is the primed phosphorylated site of substrate and X can be any residue while S is the site of phosphorylation by GSK-3 [84]. It was demonstrated that primed phosphate group forms hydrogen bonding and electrostatic interactions with triad of Arg96, Arg180 and Lys205, resulting in the optimal orientation of the N and C terminal domains of GSK-3 [85, 86]. Mutation of Arg96 impairs phosphorylation of many primed substrates by GSK-3 [36, 80, 87]. Besides requiring primed phosphate and SXXXS(p) sequence [88], substrate recognition interaction of GSK-3 also requires assistance of Phe67, Gln89 and Asn95 [85], which promote optimal positioning of substrates within the substrate binding pocket leading to better interactions and its specificity [85]. Several substrates of GSK-3 like Glycogen synthase, ATP citrate lyase, eukaryotic initiation factor 2B (elF2B) and catenin, require prephosphorylation while some do not require priming phosphorylation (cyclin D3, axin and APC) [89-96]. It has been observed that phosphorylation of primed substrate is more efficiently done as compared to non-primed substrate at least under in vitro conditions [87, 96, 97]. There is a large variability in the way kinases recognize their substrate [98]. CDK-2 recognizes canonical motif of (S/T)PX(K/R) where S/T is the P-site(where the phosphorylation has to take place) and X is any amino acid. CDK-2 also has the specificity for Pro of P+1 site because of hydrogen bonding properties of Proline containing peptides. CDK-2 also prefers a basic residue like His, Arg at P+3 position of substrate to interact with Thr160 (pTHR160) in the kinase [99]. In case of protein kinase this canonical motif is RRX(S/T), where is hydrophobic and ‘R’ is the basic residues at P+3 and P+2 sites, these basic residues interact with Glu127 and Glu230 of PKA. In addition, the hydrophobic residue at P+1 (X) position fits into pocket lined by Leu198 and Leu205 residues [100]. Another unique variation in case of GSK-3 is the preference of prephosphorylated substrate while this preference is not observed in other kinases [101]. This variability of the substrate binding site has been exploited in the design of peptides which can inhibit the enzymes specifically [102], details of which will be discussed in the next section.


4. REGULATION OF GSK-3 GSK-3 is active under resting stage and its implication in various signalling pathways, makes its regulation very important for its coordinated functioning and related physiological phenomenon. To accomplish this, GSK-3 is regulated at multiple levels mainly through, phosphorylation (Ser9 and Tyr216 in isoform). The phosphorylation of residue Ser9 results in the inhibition of GSK3 [103]. This phosphorylated serine residue at the N terminal domain in the respective isoforms acts as a pseudo-substrate for catalytic triad (Fig. 6) and blocks access of the substrate to the substrate binding site [79, 80, 104, 105]. This mechanism of inactivation mediated by Akt/PKB [106] (protein kinase B) during insulin signalling leads to the activation of glycogen synthase [107-109]. Besides Akt/PKB, phosphorylation of Ser9 can also be accomplished by other kinases like AGC kinase p70, ribosomal kinase, S6 kinase. Phosphorylation of Tyr216 [104, 110] increases the catalytic activity of the GSK-3 up to 100 fold [111]. The phosphorylation of tyrosine makes it negatively charged and facilitates the subsequent electrostatic interaction with positively charged Arg220 and Arg223 (Fig. 7). The phosphorylated tyrosine swings from its original position to a position where it facilitates the access of substrates towards the substrate binding site. Moreover, this electrostatic interaction causes the widening of the ATP binding groove for better orientation of phosphate group of ATP and the residue of the substrate to be phosphorylated [10, 79, 80]. Thus, the phosphorylation of Tyr216 in GSK-3 directs the side-chain relative to the active site, but does not cause any major change in the conformation of


residues around activation loop, as is the case observed with other kinases [89, 104]. Inactivation of GSK-3 due to phosphorylation of Thr390 by p38 MAPK has also been reported by Thornton et al. [112]. More importantly this inactivation is not reported for GSK3 since this residue is not conserved between the two isoforms [112]. It can be suggested that besides the Ser9 mediated regulation of GSK-3, this could be another alternate mechanism for selective inhibition of GSK-3 especially in the brain [112]. Understanding the mechanism of regulation of GSK-3 can help in designing new leads for its specific inhibition. Till now the regulatory mechanism of Ser9 phosphorylation has been exploited for the design of GSK-3 inhibitors. The thiadizolidinone (TDZD) derivatives were reported as inhibitors which can bind to the catalytic triad [113], which were designed based on the idea derived from the fact that phosphorylated Ser9 residue acts as pseudo substrate and inhibits GSK-3. There are many other mechanisms for which details are available at the biological level. All these mechanisms can be explored for inhibitor development but currently limited work is being taken up in the modulation of regulatory mechanism because the molecular level details are not yet available. 5. INHIBITORS OF GSK-3 As GSK-3 is currently among the most important therapeutic targets, development of its selective inhibitors is the thrust area in many laboratories across the world and several reports related to GSK-3 inhibitors have appeared. Inhibition can be accomplished through various mechanisms which can be classified into (i) inhibi-

Fig. (6). ATP binding site and catalytic triad formed Arg96, Arg180 and Lys205 with phosphate ion; PDB code-1UV5.

Fig. (7). Phosphotyrosine bound to Arg220 and Arg223 PDB code-3F7Z.

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tion of metal, (ii) inhibition at the ATP binding site, (iii) inhibition at the non–ATP binding site, (iv) irreversible inhibitors of GSK-3, (v) peptide like inhibitors, (vi) allosteric inhibitors, etc. 5.1. Inhibition by Metals GSK-3, like most other kinases, requires Mg2+ for binding of the ATP to its pocket. Lithium (Li+) salts were first to be reported as inhibitors of GSK-3. Lithium not only inhibits GSK-3 by directly competing with a magnesium ion but also acts indirectly by increasing inhibitory phosphorylation of Ser9 [114]. Lithium also carries well-known property of mood stabilization and has helped in the treatment of manic patients by inhibiting inositol monophosphatase. Inhibition of GSK-3 has also been implicated behind mood stabilizing property of lithium [115, 116]. Zinc salts have also been reported to inhibit GSK-3 by competing with the Mg2+ ions [23, 67, 117]. Ekin et al. reported an organoruthenium complex inhibiting GSK-3 with IC50 values in the picomolar range [118, 119]. However, the non-selective nature of these metal ions in the inhibition of GSK-3 has led to a decrease in the development of metal based inhibitors and is of only historical interest. 5.2. Inhibition at ATP Binding Site The ATP competitive mechanism was extensively pursued towards the development of GSK-3 inhibitors. The intensive research in the design and development of GSK-3 inhibitors led to the appearance of several classes of ATP competitive inhibitors, some of which have shown the therapeutic action in other areas. This is exactly the case with first generation of GSK-3 inhibitors such as hymenialdisine (1) [46], paullone (2) [54, 55], indirubin (3) [53] and staurosporine (4) [44, 50]. Though these molecules were not selective in nature [7], they provided important insights for the design and development of the second generation of GSK-3 inhibitors which showed fair to high selectivity [7]. Second generation GSK-3 inhibitors were a result of research studies subjected to the initially discovered synthetic small molecules and natural products [7]. The second generation of GSK-3 inhibitors includes molecules belonging to the class of heteroaryl-pyrazolo[3,4-b]pyridine (5) [48], 3-(7-azaindolyl)-4-arylmaleimides (6) [51], 1-(4-aminofurazan-3-yl)-5-dialkylaminoethyl-1H-[1,2,3]triazole-4-carboxylic acid (7) [52] and 3-anilino-4-arylmaleimides (8) [49]. Among these, CHIR-98023 (9) [45] was claimed to show high selectivity for GSK-3 over other phylogenetically related kinases. Though second generation ATP competitive GSK-3 inhibitors showed fair to high selectivity with a significant amount of activity, none of them have reached to the clinical stage. The recently reported new scaffolds of GSK-3 inhibitors include 1,3,4-oxadiazoles (10) [120122], benzo[e]isoindole-1,3-diones (11) [123], phenylmethylenehydantoins (12) [124, 125], dipyrrolo, furopyrrolopyrazinones (13) [126] and pyrazine [127] derivatives. Of these, only oxadiazoles, phenylmethylenehydantoin derivatives and pyrazine derivatives showed reasonably good activity with selectivity while others were fairly active and selective. Pyrazinones are moderately active but are non-selective in nature. The existence of some selective GSK3 small molecules in the new generation of inhibitors promises the opportunity for better approaches of understanding the features required for the design of highly selective GSK-3 inhibitors. 5.3. Inhibition by Binding at Catalytic Triad In an effort to solve the problem of selectivity, a set of thiadiazolidinone (TDZD) derivatives (14) were developed which do not compete with ATP [128]. With the help of mapping and molecular docking studies, Martinez et al. showed that this class of non-ATP competitive inhibitors may bind to the catalytic triad preventing the proper orientation of the substrate in the substrate binding site [113]. Castro et al. [129] studied the structure activity relationship for TDZD. The basic skeleton 1,2,4-thiadiazole with one carbonyl

Arfeen and Bharatam

group was kept while modifications were made at the 3rd and 5th position respectively to generate molecules with (15), (16) and (17) scaffolds. Two more class of molecules (18) (19) were synthesized by substituting the carbonyl groups at 3rd and 5th position with amino, alkyl or aryl groups. The two strategies resulted in the synthesis of large sets of thiadiazole and thiadiazolidine derivatives and were evaluated for GSK-3 inhibitory activity. Members of 5amino-3-oxo-2,3-dihydro-1,2,4-thiadiazole (17) showed activity between 6.5 and 40 M except the benzoylic derivatives which showed complete loss of activity. For other molecules of this series, decrease in the size of the substituent at position 2 showed a significant increase of activity while the absence of the hydrophobic group at position 5 showed a decrease in inhibitory activity. The quest for other members in this class prompted Martinez et al. [113] to synthesize various derivatives of rhodanines, thiazolidines, dithiazolidiones, triazolidinediones, hydantoins, which when evaluated for GSK-3 inhibitory activity showed that compounds with the aromatic substitution at nitrogen atom were most potent while activity was completely lost in case of triazolidindione and hydantoin compounds. This suggested the crucial role of S in the TDZD scaffolds in modulating the activity of GSK-3. Among the molecules which can have the possibility of binding to the catalytic triad, thiadiazolidinones are the only reported inhibitors so far, but this postulation still needs to be verified [130, 131]. 5.4. Substrate Competitive Inhibitors Hamann et al. [132] reported the semi-synthetic analogues of manzamine A (20), an alkaloid isolated from Indonesian sponge Acanthostrongylophora [133-137] as a new class of GSK-3 and CDK-5 inhibitors which can be exploited in targeting Alzheimer’s. The enzyme kinetic studies showed that these analogues inhibit GSK-3 in a non ATP competitive manner. With the help of molecular modelling studies it was predicted, that these molecules bind in the vicinity of catalytic triad. The structure of manzamine consists of -carboline heterocycle attached to a novel pentacyclic diamine core containing both eight- and thirteen membered rings on a pyrrolo[2,3-i]isoquinoline framework. To identify the pharmacophore responsible for GSK-3 inhibition, -carboline and ircinal, (precursors of manzamine) were tested and were found to be inactive, indicating that the whole manzamine moiety is responsible for GSK-3 inhibition. SAR studies showed that replacement of hydrogen at carboline 6 or 8 position with OH, OMe, OEt, and OTs group did not affect the inhibitory activity except hydroxyl derivatives which were slightly more active [138]. Substitution of N9 atom of the carboline heterocycle with small groups like Me, Et, did not show any significant effect on activity, while substitution with larger groups like i-But, or t-BuOCOMe led to inactive compounds. Removal of unsaturation from an eight membered ring of manzamine resulted in a slight increase in activity. The absence of double bond between positions 15 and 16 resulted in inactive molecules. Substitution of carbonyl group at position C31 led to less potent manzamine derivative. The inclusion of epoxy function in cyclooctane ring did not affect the activity. A very recent addition to the substrate competitive inhibitors was 5-imino-1,2,4-thiadiazole derivatives (21) (ITDZD), reported by Palomo et al. [130], which inhibited GSK-3 in low micro molar range. The enzyme kinetic study showed that these inhibitors bind to the GSK-3 in reversible non-ATP competitive and substrate competitive manner and have been claimed to be the first small molecule which inhibits GSK-3 by competing with substrate. 5.5. Irreversible Inhibitors Conde and coworkers proposed a new approach for the selective inhibition of GSK-3 based on the hypothesis that Cys199 located in the ATP binding site of GSK-3 [139, 140], replaced by other amino acids in CDK’s and other homologous kinases can irreversibly be modified with the help of organic molecules bearing


Fig. (8). Inhibitors of ATP binding site.


7


Arfeen and Bharatam

Fig. (9). TDZD and its modifications at 3rd and 5th positions of 1,2,4-thiadiazole ring.

[141]. Molecular docking studies using rDock software were performed to predict the binding pose of irreversible inhibitors in which the distance was found to be 4 Å between the inhibitors C atom and S atom of Cys199. The model reactions were also carried out using mercaptoethanol and phenyl -halo methyl ketones to model the reactivity of Cys199 in which the S-alkylated products were obtained in major amount [141].

Fig. (10). Substrate competitive inhibitors (manazamine and ITDZD).

-halomethyl group. These molecules were initially identified during random experimental exercise, from which compound 2chloroacetyl-4,5-dichlorothiophene showed GSK-3 inhibition at lowest concentrations [139]. A Second collection of 15 molecules bearing common structural feature (-carbonylthienyl (22) or phenyl (23) were further tested in the second phase [139]. It was observed that compounds bearing the group chloromethylhalothienyl ketones showed good activity. From the results it was also clear that besides the presence of acetyl group, presence of electron withdrawing substituent at carbon to carbonyl group enhances activity in this series of compounds. Singly substituted derivatives showed optimum results for GSK-3 inhibition while diacetyl derivatives did not show any improvement over the monoacetyl counterpart. The mechanism for GSK-3 inhibition was also investigated which was claimed to bind in a non-ATP competitive manner [139]. Perez et al. performed a series of studies to establish the reactivity at Cys199 [141]. The classical inhibitors belonging to the class of adenine, benzimidazole and maleimide were chemically modified to their respective -halomethylketone and acetyl derivatives. The modified derivatives so obtained were evaluated as GSK3 inhibitors and it was found that -halomethylketone derivatives were more potent than their acetyl counterparts. The covalent modification was established biophysically using MALDI-TOF. Spectral analysis showed the mass corresponding to GSK-3 when reversible inhibitors were used while in the case of irreversible inhibitors, mass corresponding to GSK-3 and its inhibitors were present

Fig. (11). First Irreversible inhibitors of GSK-3.

5.6. Peptide-like Inhibitors After two decades of intense research on the ATP binding site of GSK-3, design of selective ATP competitive inhibitor is still a problem, so design of such molecules which can compete with substrate to prevent its binding was considered as an alternate strategy. Several small phosphopeptides derived from the amino-terminal end of GSK-3 appeared over last two decades (based on the mechanism of phosphorylation at the Ser9/21 resulting it to act as pseudosubstrate, thus competing with primed substrate for binding to the substrate binding site). One such phosphopeptide is Thr-ThrpSer-phe-Ala-Glu-Ser-Cys (24) corresponding to residues 7-14 of GSK-3 which was found to inhibit the phosphorylation by GSK-3 against phosphorylated substrates but not against nonphosphorylated substrates [87]. Bax et al. [142] reported FRATide a 39-mer peptide with the sequence corresponding to residues 188226 of GSK-3. This peptide forms two -helices and binds to the C-Terminal lobe adjacent to the substrate access channel but not obstructing it. FRATide competes with Axin for binding to the GSK-3 and inhibited the phosphorylation of Axin and -catenin but it does not inhibit the GSK-3 activity towards peptide derived from elF2 or Glycogen Synthase. Plotkin et al. [143] reported 11 amino acid phosphopeptide L803 and L803-motos (25), derived from the heat shock factor-1 protein (HSF-1) with following sequence (KEAPPAPPQS(p)P), which inhibited the phosphorylation of GSK-3 targets such as Glycogen Synthase, IRS-1 and -catenin. 5.7. Inhibition by Binding at Allosteric Site Palomo et al. [144] identified compound VP0.7 (26) which showed the IC50 value in M. The enzyme kinetic assays showed that this particular molecule binds in non-ATP as well as in nonsubstrate competitive nature; suggesting the allosteric modulation of GSK-3. The docking studies with the help of Autodock software along with fpocket analysis indicated that the probable binding pocket for the VP0.7 is formed of Arg209, Thr235, Ser236, Thr330, Pro331 and Leu169. An appreciable difference was observed in the A loop of the substrate binding site in the docked complex as compared to the original structure [144]. Along with TDZD, ITDZD and manzamine alkaloids, peptide inhibitors can be grouped into substrate competitive inhibitors. To the best of our knowledge out of the several molecules reported, only one belonging to the TDZD class has reached the clinical trials. The substrate competitive inhibitors, often have been overlooked in the high-throughput screening experiments due to the weak binding interactions with enzyme [145]. Another problem with these types of inhibitors is in vitro assays, as their concentration is higher under in vitro conditions than physiological concentration, the in vitro assays do not reflect the true potency of sub-



9

Fig. (12). Structure of phosphopeptide reported from the N-terninal end of GSK-3 (residues 7-14) (above) and L803-mts (below).

Fig. (13). Structure of VP0.7 (Allosteric modulator).

strate competitive inhibitors, and in many cases these inhibitors show better efficacy in the cell based assays rather than in the cell free assays [102]. Weak binding interactions made by this type of inhibitors with GSK-3 have been proven to be disadvantageous for them. 6. STRUCTURAL INSIGHTS FROM SELECTIVE GSK-3 INHIBITORS The ATP competitive strategy is one of the most exploited strategies for the inhibition of protein kinases and as a result, many ATP competitive inhibitors for GSK-3 have been reported. In terms of selectivity, best among them is AR-A014418 [146], (claimed to be most selective or almost specific). Other inhibitors belonging to the same league are 1-azakenpaullone [47, 54], aminopyrimidine derivatives [147], arylindolylmaleimide (SB216763) [148] and anilinomaleimides [148], while in recent ones oxadiazoles derivatives [120, 121] showed promising results. With the emergence of these reportedly selective inhibitors, probes of understanding various factors responsible for selectivity also increased and as a result several explanations were offered. Two factors were stated to be majorly responsible for improved selectivity (i) the

interactions with amino acids that are unique to GSK-3 active site (selectivity residues) [60] and (ii) the larger size of the ATP binding cavity in GSK-3 when compared to its homologous counterparts [58, 149]. The residues identified for selectivity in GSK-3 are Leu132 (Phe80 in CDK-2, CDK-3 and CDK-7), Tyr134 (Phe82 in CDK-2), Arg141 (lys89 in CDK-2) and Cys199 [58, 60, 146]. Leu132 and Cys199 form the part of the hydrophobic pocket in GSK-3 [60]. The other residues making the hydrophobic pocket are Leu130, Val10, Met101, and Phe201 [60]. This hydrophobic pocket in GSK-3 is larger, flatter and accessible for the bulkier groups, as compared to the hydrophobic pocket of other related protein kinases. The reason for this extra space in GSK-3 is the presence of smaller Leu132 in place of Phe80 in CDK-2, CDK-3 and CDK-7 [58, 149]. Kozikowski et al. [60] designed and synthesized compounds (27) and (28) etc. [150] and identified 3-indolyl-4-indazolylmaleimide class of compounds (29) as potent and selective inhibitors. They further observed that benzofuranyl bearing maleimides (30) showed good inhibitory activity, but were non-selective in nature. Utilizing above mentioned structural features of GSK-3, series of compounds were designed and synthesized, which when evaluated against a panel of 22 kinases showed good results of potency and improved selectivity. The compounds (27) and (28) showed the best results of potency and selectivity. Meijer et al. [53] reported the crystal structures of the indirubin-3-monoxime (31) and its bromoderivative, which was found to be 16 folds more selective for GSK-3 against CDK-2. Polychronopoulous et al. [58] investigated the role of substitution at 5th and 6th positions of indirubin. The substituted indirubins were designed, synthesized and modelled using crystal structures of indirubin with GSK-3, CDK-2 and CDK5/p25. With the help of modelling studies the authors attributed the selectivity of 6-bromo derivatives to


Arfeen and Bharatam

Fig. (14). Indazole benzofuran derivatives of maleimides and thiazole-methoxybenzyl-thiourea and pyrazine derivatives.

the absence of the bulkier phenyl group (presence of Leu132) in GSK-3 when compared to CDK-2 (Phe80). The same was observed with the thiazole-methoxybenzyl-thiourea (32) in which it was found that thiazole-nitro substituent is placed adjacent to the Leu-132 [146]. Analysis of the SARs and structural studies with 5arylpyrazolopyridazines [48] revealed similar picture where the fluorine atom of the compound is comfortably placed in the surroundings of Leu132 side chain and the acyl portion of the amide i.e.CH2-piperidine ethyl system occupies a similar position as the anisole system in compound (32). Another factor contributing to the larger cavity size is orientation of the Arg141 side chain, which points away from the cavity. In CDK’s Lys89 is in a similar position, points inwards and interacts with Ile10 of the glycine rich loop. Therefore, anisole system of thiazole-methoxybenzyl-thiourea in GSK-3 is able to place itself in the vicinity of Arg141 while the similar occupancy is not possible in case of CDKs [146, 149]. Another feature which contributes to larger cavity size is the orientation of Asp200 side chain, which is away from the cavity in GSK-3 while in case of CDKs the side chain of this residue is Asp which is directed towards the cavity. Bhat et al. [146] co-crystallized Ar-A014418 with GSK-3 as well as with CDK-2 to understand the structural basis for selectivity. They identified that the presence of salt bridge formed by Glu137 and Arg141 marks the boundary of the ATP binding site in GSK-3 (Fig. 15). The equivalent residues in CDK-2 are Gln85 and Lys89 which have antiparallel orientation resulting in Lys89 to point inwards and eventually causing reduction in size of active site

but at the same time creating the wider entrance point. The authors explored that Pro136 (His84 in the case of CDK-2) may also contribute to the formation of salt bridge in GSK-3. The selectivity of AR-A014418 (32) against CDKs was partly attributed to difference in the entrance region of the ATP binding pocket. In GSK-3, phenyl group fits well in this area. The authors demonstrated that if at all this molecule binds to the CDKs, Lys89 would interfere. Another important clue obtained from the work was fitting of the planar nitrothiazole part around Cys199 area of GSK-3. All these structural details provide the necessary platform for the computational design of selective GSK-3 inhibitors. Berg et al. [127] synthesized pyrazine derivatives (33) and performed cocrystallization of the key analogues to develop the highly potent and selective inhibitors. By comparing the ATP site of CDK2 and GSK-3 in the cocrystalized structures of pyrazine derivatives, the research group revealed the difference in the ATP binding site especially around Pro136/Glu137/Thr138 in GSK-3 (His84/Gln85/Asp86 in CDK-2) and Ile62 in GSK-3 (Ile10 in CDK-2). It was claimed that the presence of Pro136 in GSK-3 leads to differences in the conformation of main chain making this area significantly larger in GSK-3 as compared to CDK-2. The cocrystallized structures further showed that phenyl ring of compound (34) is in non-planar arrangement with respect to pyrazine ring (dihederal angle of +21 in GSK-3 and -13 in CDK2). The authors attributed the selectivity of the pyrazine derivatives to this dihederal angle as the selectivity in the series increased as the dihederal angle between the pyrazine and the phenyl ring increased due to substitution at R1 position.



11

Fig. (15). Hydrophobic pocket of GSK-3 in ATP binding site (shown in cyan colour); PDB code-1Q5K.

7. STRATEGIES FOR DESIGN OF INHIBITORS Besides crystal structure based studies (which have been discussed earlier), several molecular modelling studies were performed to model the pharmacophoric features of GSK-3 as well as its ligands. The molecular modelling studies have contributed to the understanding of requirements for selective inhibition of GSK-3 in recent years. These molecular modelling studies can be grouped into (i) QSAR analysis (ii) molecular docking, pharmacophore mapping, virtual screening and (iii) molecular dynamic simulation studies. In this part of review, we present various computational efforts made to get the insights of ligand binding sites and to design selective GSK-3 inhibitors; importance is given to molecular modelling studies which are supported by experimental evaluation. 7.1. QSAR Analysis QSAR provides a direct method to correlate the in vitro / in vivo pharmacological data to the structures of a series of ligands and helps in understanding the pharmacophoric features required for the therapeutic action. This is a method of choice when the biological activity for a series of molecules is numerically expressed (ex. IC50). Several efforts have been made using QSAR to correlate the structural descriptors to the GSK-3 therapeutic action [113, 151159]. 3D QSAR studies have been carried out for several classes of GSK-3 inhibitors. These include QSAR studies on (i) paullones [156], (ii) 3-anilino-4-arylmaleimides [158], (iii) thiadiazolidinones [113], (iv) pyrazolopyrimidine [157], (v) pyridazines [159], and (iv) pyrazolopyridazines [160]. All the studies are based on grid based 3D-QSAR methodologies using CoMFA and CoMSIA algorithms; details of selected publications are reviewed in the following paragraphs. Patel et al. [159] performed a CoMFA analysis on pyrazolo[3,4-b]pyrid[az]ine (35) derivatives and identified various key spots for this class, modulation of which help the molecules to attain selectivity against related kinases. A CoMFA model was obtained by using a dataset of 59 molecules (selective and nonselective inhibitors) and validated using a test set of 14 molecules. The best model was obtained by applying leave-one-out (LOO) cross-validation with r2cv and conventional r2conv values of 0.60 and 0.97, respectively. This model explained (i) the observed variance in activity and (ii) the structural differences between the selective and non-selective pyridazine derivatives. The contour map analysis showed that substitution at 3rd position (Fig. 16) with electropositive groups of optimal steric bulkiness improves the activity. Substitution at 5th position is very crucial for GSK-3 potency as groups

with electropositive character and electron withdrawing nature can improve potency. Lipophilic substitution at the 5th position also leads to improved potency. Substitution at 6th position with electronegative or lipophilic groups is favored and leads to improved binding affinity and selectivity. The 3rd and 5th positions are critical for potency while the 6th position is important for selectivity. Dessalew et al. [157] performed CoMFA and CoMSIA analysis on pyrazolopyrimidine (36) derivatives using training set of 49 molecules. Best models were generated by applying LOO crossvalidation study with, r2cv values of 0.53 and 0.48 for CoMFA and CoMSIA, respectively. The models were validated using a test set of 12 molecules with predictive correlation coefficients (r 2pred) of 0.47 and 0.48, respectively. This study showed that the structure of the pyrazolopyrimidine derivatives can be divided into region A, (aromatic substituent at N1 atom of adenine ring), and central core (formed by adenine ring), substituent of the hydrazone nitrogen can again be divided into region B (the group containing heteroatom) and C (extended side chain) (Fig. 16). Presence of aromatic bulkier substitution at N1 atom of adenine ring is important for the activity and constitutes region A, the best active molecules had the benzimidazole as the substituent in the region A. CoMSIA model suggested that the conformation of the substituent on benzimidazole is important as the substitution of the hydrophobic group on the imidazole ring results in an increase of activity while the same substitution on benzene ring is unfavourable. Presence of heteroatom in region B is necessary for the activity; substitution of bulky group at the third position of pyrazolopyrimidine ring is disfavorable but the substitution of electropositive groups at the same position results in an increase of binding the inhibitor to protein, without affecting the activity. The Presence of the hydrophilic group with optimum steric effect increases the activity of molecules while the extended side chain in the region C results in the decrease of activity. Based on the above mentioned pharmacophoric features, new ligands were designed which when docked using FlexX, showed all major interactions of hinge region (Asp133 and Val135) and electrostatic interactions with Arg141. The newly designed ligands also showed interactions with Thr138 through the bridged water molecule, which has been claimed to be responsible for selectivity [83]. Lescot et al. [158] correlated the three dimensional structure of 3-anilino-4-arylmaleimides (37) with their biological activity by 3D-QSAR (CoMFA) studies and compared the results with crystallographic data of GSK-3. The model so obtained was not in correspondance with the interaction map obtained from the active site but provided some important features in relationship with GSK-3ligand interactions. 74 molecules of 3-anilino-4-arylmaleimide


Arfeen and Bharatam

Fig. (16). Basic scaffolds used in the QSAR docking study, Pyrazolo[3,4-b]pyrid[az]ine, Pyrazolopyrimidine, 3-anilino-4-arylmaleimides.

series were used to build the model. The basic scaffold of 3-anilino4-arylmaleimide can easily be divided into three regions A, B and C (Fig. 16). Region A corresponds to the NH group of 3-Anilino fragment, substitution of this nitrogen results in a loss of activity showing importance of secondary amine at this position. Region B corresponds to 3rd, 4th and 5th carbon atoms of the benzene ring of the 3-anilino fragment which is occupied not only by the hydrophilic groups such as OH or COOH but also by the hydrophobic groups like chlorine. The substitution of this region with the bulkier groups like 3,5-dichloro-4-hydroxy compared to 3-carboxy-4chloro or 3-chloro-4-hydroxy resulted in increased biological activity. Region C belongs to 4th position of phenyl group. The substitution of the nitro group at this position improves the affinity when compared with chloro and methoxy substituted derivatives. Maleimide ring forms hydrogen bond with two residues (Asp133Tyr134) which are responsible for its affinity. Nitrogen of aniline binds to the Val135 (distance was 3.5Å) which when substituted with a methyl group showed decrease in activity. Polar groups in the region B lead to strong interaction with residues Arg141 and Gln185. Fang et al. [161] developed a new protocol (Fig. 17) for predicting novel GSK-3 ATP competitive inhibitors. This protocol includes a combination of 3D QSAR methods and structure based docking methods. This protocol included the following steps (i) development of 3D QSAR models, (ii) selection of additional compounds based on the substructure search for the maleimide core, (iii) use of FlexX docking program for virtual screening, (iv) prediction of the GSK-3 inhibitors of the virtual hits by 3D QSAR model, and (v) the confirmation of the top scoring compound with experimental results. 3D QSAR model was generated using a data set of 38 benzofuran-3-yl-(indol-3-yl)maleimide derivatives (30 in the training set and 8 in the test set). Based on the substructure search of maleimide, 28826 molecules were identified from the pubchem database. These identified molecules when subjected to Lipinski’s rule of five, resulted in 10429 hits. These filtered molecules, when subjected to molecular docking using the FlexX docking program, resulted in 617 virtual hits. Among the 617 virtual hits QSAR model identified 93 hits. Out of these 93 compounds, 23 compounds showed the IC50 value in the range of 1.3 to 480 nM. The experimental results showed the predictive ability of this protocol.

Fig. (17). Protocol of combined ligand and structure based approaches.

In the past 3D-QSAR studies on GSK-3 inhibitors were mainly utilized for lead optimization so as to develop the new molecules that can have improved biological activity. The recent efforts based on 3D-QSAR made by Fang et al. focused on the identification of new leads utilizing the clues obtained from the previously reported inhibitors. It is only one such example of its


kind. Clearly there is a wide scope for this type of efforts in future; especially when the results from QSAR are further taken to experimental validation, the applicability becomes quite relevant. 7.2. Molecular Docking, Pharmacophore Mapping, and Virtual Screening Virtual screening is an important pharmacoinformatic tool [162], which helps in the design of new ligands. Several technologies are associated with the virtual screening approach. These include molecular docking, pharmacophore mapping [61, 163], fragment based approaches [62], etc. In addition, to the imposition of empirical rules based on conformational flexibility, log P values, molecular weight, numbers of hydrogen bond donor and acceptor atoms, etc. parameters are important. Among these, molecular docking technology can independently be used to obtain the structural details of enzyme inhibitor interactions. However, this technology also finds extensive applications in virtual screening. Pharmacophore methodology employs the mathematical tools associated with mapping algebra and helps in developing models which can be routinely applied for virtual screening after an appropriate validation. Employing these technologies, a protocol can be developed for successful screening of a virtual library of compounds, which can be suggested for experimental drug discovery efforts. Many groups have employed such strategy in the recent past for the identification of selective GSK-3 inhibitors [59, 62, 124, 125, 160, 163-166]. These efforts adopts varying strategies with the common goal of identifying new ligands. Given below is a review of these virtual screening strategies which are linked with experimental validation. Fig. (18) shows a schematic representation of steps followed during virtual screening.

Fig. (18). Schematic representations of the steps adopted in the virtual screening.


13

Polgar et al. [166] developed a virtual screening protocol using molecular docking approach to distinguish potential GSK-3 ATP inhibitors from a collection of virtual compounds. Experimental high-throughput screening analysis was also performed to establish the effectiveness of the protocol. Protein structures with PDB code 1Q4L, 1UV5 and 1Q3D were selected for virtual screening of the compounds using FlexX, FlexX-Pharm and FlexE software. Enrichment factor, a parameter which assesses the quality of rankings was employed with FlexX and FlexX-Pharm based molecular docking analysis. The authors showed that the torsional energy based pharmacophoric constraints can be applied successfully for screening GSK-3 kinase ATP competitive inhibitors. The screening protocol was able to pick four out of six classes of compounds that were identified in experimental high throughput screening [166]. The virtual screening protocol also picked a large number of false positives and false negatives which indicated its significant limitations. The Inaccuracy of the docking score, application of pharmacophore constraints and the neglected flexibility of the active site were held responsible for the above mentioned limitations. Khanfar et al. developed a pharmacophore model using the most active marine derived phenylmethylene hydantoins [124], with the help of distance comparison technique (DISCOtech ) module implemented in sybyl8.0 software package. The model included the important interactions known for GSK-3 inhibitors binding at ATP site. The main pharmacophoric features identified were one hydrogen bond donor (HBD) and two hydrogen bond acceptor (HBA) and two hydrophobic sites [125]. The model was validated using an in-house database which included 200 active and inactive molecules. This pharmacophore was utilized for screening in-house as well as NCI and Maybridge databases which resulted in the identification of 569 out of 289903 hits which when subjected to the Lipinski rule of 5 (for drug like characters) resulted in 53 hits. These identified 53 molecules were docked into the active site using GOLD and Surflex-Dock software packages resulted in 34 compounds showing proper conformations and expected interactions. During docking study only those molecules were retained (i) which showed at least three out of four H-bond interactions with important amino acids (Val135, Asp133, Arg141, Gln185) (ii) retained orientation similar to one or more of the reported inhibitors and (iii) binding score equal to or higher than the cocrystallized GSK-3 inhibitors. These 34 compounds were assayed in vitro using the Invitrogen Z’LYTE Kinase assay kit. Out of which 14 hits showed promising results [125]. Two compounds that showed high potency with good selectivity towards GSK-3 over CDK-2 were evaluated in vivo for enhanced hepatic glycogen and brain permeability. This resulted in the identification of 2-anilino-5-phenyl1,3,4-oxadiazole analogue (38) as the promising lead for GSK-3 inhibition with good brain permeability. 32 Derivatives of this identified lead were synthesized by the same research group, to study the structure activity relationship. Taha et al. [160] employed the HYPOGEN module of the CATALYST software package to construct the pharmacophore map for GSK-3 inhibitors. 152 inhibitors belonging to the classes of anilinomaleimides, indolyl maleimide and pyrazolopyridines were employed for the generation of possible binding modes within the GSK-3ß ATP binding pocket. The pharmacophoric space was explored under reasonably imposed “boundaries” via six HYPOGEN run and selected two subsets of training compounds. Training molecules were selected in such a way as to cover the range from most active molecule to the least active molecules. 3D Diversity of the most active molecules was given more emphasis in each training subset. HYPOGEN was restricted to explore the pharmacophoric models incorporating from zero to three features of any particular selected feature type (i.e. HBA, HBD, hydrophobic and Aromatic ring). HYPOGEN was also restricted to explore only four and five featured pharmacophores. This constrained methodology in the development of pharmacophore resulted in the generation of


Arfeen and Bharatam

Fig. (19). Structure of compounds identified in virtual screening 2-anilino-5-phenyl-1,3,4-oxadiazole, cimetidine, gemifloxacin and hydroxychloroquine.

the pharmacophore models rich in features of the ATP binding pocket. Subsequently a QSAR model was also generated from the same set of inhibitors as mentioned above. Genetic algorithm and multiple linear regression analyses were utilized to select the optimal contribution of pharmacophore and physiochemical descriptors, capable of explaining the diversity in bioactivity over the large set of molecules. The QSAR equation and associated pharmacophore model was then employed for virtual screening of in house 3D database of established drugs (1490 molecules) and was able to retrieve three (39, 40, 41) molecules from the structural database which showed potent GSK-3 inhibition both in vitro and in vivo. As selectivity has always been an important issue for the protein kinase inhibitors [167], other modes of inhibition has been considered as an alternative for the selective inhibition of protein kinases. One of those strategies is the inhibition through binding at the substrate binding site and the other is the modulation through allosteric binding site [168]. These allosteric modulators bind to unique regions [169] of the kinase, inducing the conformational changes, resulting in the modulation of the enzyme activity and sometimes in preventing the substrate binding [170]. This approach can result in the selective inhibition without competing with ATP. Palomo et al. [144] have utilized the pocket program to identify and typify several drugable binding sites including allosteric tempering cavities for GSK-3 inhibition. Twenty five ligand co-crystallized complexes were used for the analysis. In most of the complexes, the program was able to identify at least 10-12 different cavities while seven pockets were always found in every structure. Out of these seven cavities ATP binding sites, substrate binding site (vicinity of catalytic triad) and axin/fratide binding sites are well known and well characterized sites. These seven drugable binding sites are listed in Table 2. The results of fpocket for the above mentioned sites were established using the co-crystallized ligands obtained from protein data bank. The research group also investigated the binding site for Manzamine A alkaloid (20) using enzyme kinetic studies which included the two setsof experiments. In one set of experiments the concentration of the ATP was varied while keeping the concentration of alkaloid and substrate constant. In the second set of experiments the concentration of substrate was varied while keeping the other two constant. Double reciprocal plot indicated that the above mentioned alkaloid as non-ATP but substrate competitive inhibitor. The allosteric modulator (26) was also identified from this study. The combination of pharmacophore, molecular docking and virtual screening has always been the method of choice for identification of new leads. Recent times have seen various new approaches for the development of more reliable virtual screening protocols so as to minimize the false positives and negatives. Be-

sides identifying new leads, more efforts should be made towards the development of these identified leads into drug molecules. 7.3. Molecular Dynamics Simulation Studies Biological systems generally consist of a large number of primary (residues) and secondary (helices and sheets) structural elements; interactions among them are dynamical. The equilibrated dynamical state gets modulated due to the presence of xenobiotic. An understanding of original dynamical state of macromolecular structure and the changes induced due to the presence of drug molecules ideally would provide necessary clues for the drug action. To model this dynamical behavior, molecular dynamics (MD) simulation technology can be employed. In the MD simulation study, atoms are allowed to move for a period of time under Newtonian laws of physics, representing the dynamical state of molecules. A very few studies were taken up on GSK-3 using MD simulations. Most of these studies address the behavior of the enzyme and identify the importance of specific units of the enzyme (importance of conserved water, allosteric regulation, mutation of specific residues, dynamics of specific substrate, etc.). The microscopic changes in the dynamism due to binding of ligands were not taken up extensively. A review of the current studies on GSK-3 enzyme MD simulations is presented below [86, 171-175]. Crystal structures of enzymes with inhibitors show water molecules trapped in the active site that sometimes may play critical role in stabilizing the protein inhibitor complex. Lu et al. [171] carried out molecular dynamic simulation studies to understand the role of these conserved water molecules in GSK-3. The study was conducted on ten crystal structures (PDB codes- 1Q3D, 1Q3W, 1Q4I, 1Q4L, 1Q5K, 1ROE, 3DU8, 3F7Z, 3GB2 and 3I4B). In all the systems, water molecules are found around Thr138 and Gln185 and within the radius of 3.5Å from oxygen or nitrogen atom of inhibitors; the important water molecules were considered for MD simulation study on 1R0E. The system was fully optimized using ONIOM (our own N-layer integrated molecular orbital molecular mechanics) method implemented in Guassion03. The highest layer was treated with density functional theory (DFT) B3LYP method using the 6-31G(d) basis set, while lower layer was treated with AMBER parm96 force field. The dynamical behavior observed during ONIOM optimization, led to the conclusion that conserved water molecules have a tendency to move and orient themselves to form hydrogen bond networks. The optimized system was further analyzed in the context of the theory of Atoms In Molecule (AIM), which suggested that the conserved water molecules are capable of forming multifaceted hydrogen bonds. One of the important observations in this study was, formation of intramolecular C-H---O hydrogen bond in the ligands present in 3F7Z. Though this hydrogen bond is weak in nature it has been implicated for the observed


Table 2. ENTRY


15

Amino Acid Residues in Each of the Seven Cavities Found on GSK-3 using Fpocket Program Probable Inhibitor Binding site

Residues Surrounding The Sites Identified from Fpocket Program

1

Cavity 1 (ATP binding Site)

Lys85, Glu97, Asp113, Tyr134, Val135, Thr138, Asn186, Leu188, Cys199, Asp200

2

Cavity 2(catalytic triad)

Phe67, Gln89, Lys89, Lys94, Phe93, Asn95, Arg96, Glu97, Arg180, Lys205

3

Cavity 3 (Substrate binding site)

Ser215, Arg220, Arg223, Phe229

4

Cavity 4

Tyr140, Arg144, Arg148, Gln185, Ser219, Arg220, Tyr221, Tyr222, Glu249

5

Cavity 5

Met26, Thr38, Tyr56, Tyr71, Lys86, Ser119

6

Cavity 6

Glu80, Arg111, Arg113, Asp133, Val135, Asp190, Lys197

7

Cavity 7 (probable binding site of VP0.7)

His173, Cys178, Leu207,Arg209, Glu211, Thr235, Ther330, Tyr234, Ser236, Ser369

increase in brain penetration and pharmacological activity of the reported oxadiazole inhibitors [171]. This weak intramolecular hydrogen bond has been further held responsible for better stabilization of oxadiazole derivative in the active site where the inhibitor is favorably aligned within the GSK-3 binding pocket. The optimized structure of 1R0E (without bridged hydrogen bond), when subjected to an MD simulation run of 6 ns encountered a larger conformational change in the propanediol side chain of the inhibitor as compared to optimized systems in which the inhibitor was associated with the bridged hydrogen bond. The B- factors of C atoms showed smaller values in systems with bridged hydrogen bond as compared to the systems without bridged hydrogen bond. The study also showed that conserved water molecule gets exchanged and equilibrate with bulk solvent. Buch et al. [172] have utilized molecular dynamics approach to analyze catalytic groove, substrate binding domain and the allosteric site of GSK-3. MD simulations were performed on the following models (i) unbound GSK-3(p).ATP complex, (ii) substrate bound GSK-3(p).ATP complex, (iii) inhibitor peptide bound GSK-3(p).ATP complex, (iv) substrate bound to the allosteric site of GSK-3(p).ATP complex and (v) inhibitor peptide bound to the allosteric site of GSK-3(p).ATP complex. A Template of the regulatory domain of HSF-1 was used to construct the model for substrate peptide while template of L803 was used to model the peptide inhibitor. The first component of this study was taken up to understand the effect of phosphorylated Tyr216 on catalytic groove. The MD simulation studies on unbound GSK-3(p).ATP complex showed that, the substrate binding site opens up in case of phosphorylated Tyr216 complex and closes in case of unphosphorylated Tyr216 complex. This opening and closing of the substrate binding site is regulated by C-helix, activation loop and P loop. This molecular dynamics study also showed that in an unphosphorylated complex Arg220 interacts with gamma phosphate of the ATP and Asp181 while the Arg223 interacts with Asp200 and helps in the closing of substrate binding site. These interactions are neutralized in case of phosphorylated complex thus broadens the substrate binding site and grant full access to the substrate. The second component of this study was performed to understand the interactions made by the substrate as well as the inhibitory peptide at the substrate binding site. An electrostatic interaction was observed between the Ser10(p) of the peptides (substrate peptide and inhibitor peptide) and the catalytic triad. It was shown that amino acids (Arg96, Arg180 and Lys205) of the catalytic triad are the only amino acids which make electrostatic interactions with phosphorylated serine. The third component of this study was to identify the possible allosteric site in GSK-3. The MD simulation run of the models in which the peptides are bound to a proposed allosteric site of GSK-3(p).ATP complex, indicated less fluctuations in the activation loop, C helix and P loop. The motion correlation along the

simulation also showed that binding of these peptides to Allosteric binding site shifts the kinase from inactive to active conformation. The molecular dynamic simulation studies on the above mentioned allosteric bound models further showed the formation of salt bridge (between Lys85 and Glu97), bringing the C helix closer to P-loop and activation loop causing the stabilization of the substrate binding site. Lu et al. [86] performed the MD simulation studies to understand the basis of specificity of GSK-3 for the primed substrate and to answer the question why it specifically phosphorylates at P+4 site. The models of GSK-3.ATP.(p)GS and GSK-3.ATP.GS complexes (GS = Glycogen Synthase) were constructed and subjected to a simulation period of 12 ns using the AMBER software package. Analysis of RMSD values showed a large difference between the backbone C atom of the complexes bound to substrate and unphosphorylated substrate bound complex with larger values in later case, indicating the conformational change. The B-factor analysis indicated a better correlation of experimental flexibility and the predicted flexibility for primed substrate bound complex than unprimed substrate bound complex. The B - factor analysis also showed that the change in conformation of the activation loop is not affected by the primed or unprimed states of the substrate. The superimposition of the crystal structure and the simulated structure showed the presence of closed conformation of C-loop and turn secondary structure of the A loop in case of complex bound with primed substrate, while the open conformation for the same was observed for unprimed substrate bound complex. The electrostatic potential of catalytic triad was calculated for both the models using the Delphi program and the potential was found higher in case of GSK-3.ATP.(p)GS as compared GSK-3.ATP.GS. This indicates the need for negatively charged primed substrate capable of effectively neutralizing the positive charge that is clustered over three residues of catalytic triad. The neutralization of this positive charge by primed phosphate of the substrate helps in proper alignment of N-terminal and C-terminal lobes. It was also shown that the occupancy of the catalytic triad with the hydroxyl group containing serine does not effectively compensate for the positive charge and leads to deformation of catalytic triad. It was further observed that the distance between the reaction centers of the ATP and the residue of the primed substrate to be phosphorylated is shorter as compared to the unprimed substrate. Moreover MM-GBSA calculations and decomposition of total energy per residue further showed a favorable site of interaction between the residues of catalytic triad and the primed substrate [86]. These results explained the presence of a P+4 primed phosphorylation specificity of GSK-3 for its substrate. Lys85 of GSK-3 binds with ATP at a location between the and phosphate of ATP and is one of the conserved residues present in the ATP binding site. It forms a salt bridge with Glu97 and


forms hydrogen bond with oxygen atoms attached to the and phosphate of the ATP respectively. It plays an important role during phosphorylation [176], and shows hydrophobic interactions with nitro group of alsterpaullone and chlorine group of the 3-anilino-4arylmaleimide[174], loss of activity due to Lys85 mutation has also been reported [174, 177]. Sun et al. [174] investigated the importance of Lys85 by performing the MD simulation studies on the wild type GSK-3 and mutated GSK-3 (K85R) for 5 ns using the molecular mechanic force field. During the simulation studies it was observed that mutated Arg85 could form only one hydrogen bond with the ATP instead of two, as was the case with Lys85. The salt bridge also vanished in the mutated model, causing the movement of Arg96 away from its original position, followed by distortion of catalytic triad and more open conformation of the substrate binding site. Loss of salt bridge also resulted in the loss of hydrogen bond interaction of Glu211 with Lys205 leading to the movement of Glu211 side chain out of the substrate binding groove and affecting the substrate binding. In case of wild type GSK-3, a phenyl ring of the Phe93 is parallel to the substrate binding groove while in case of mutated GSK-3 the phenyl ring orients itself in a way to block access to the substrate binding groove. The MM-GBSA calculation showed that the electrostatic interactions are stronger in the wild type model as compared to the mutant model while the total binding energy is lower for the wild type model as compared to the mutant model. The MD simulations were carried out to understand the basis of selectivity for ATP competitive inhibitors. Chen et al. [173] provides an insight into the factors responsible for the selective inhibition of GSK-3 by 1-azakenpaullone (42) as compared to the 2azakenpaullone (43) and alsterpaullone (44). The MD simulations were performed on the complexes of the above mentioned inhibitors with the GSK-3 as well as CDK-5. The system was set using the Amber03 force field. The MD simulation results indicated that the interaction between the paullones and Val135 in GSK-3 is stronger than the interaction between the paullones and Cys83 of CDK-5. Though this differentiated affinity contributes to the selectivity, its affect is very limited. It was also stated that the substitution at the position 9 of the 1-azakenpaullone and replacement of the C atom at the position 2 with the N atom (2-azakenpaullone) might affect the potency of inhibitor but not the selectivity. The authors stated that the most effective way of improving the selectivity is to change the interaction with the Asp86 in CDK-5 as was the case with 1-azakenpaullone. Hymenialdisine (45) and dibromocantharelline (46) were isolated from the marine sponge showed different patterns of inhibitory activity. Dibromocantharelline, moderately but selectively inhibits GSK-3 activity. Zhang et al. [175] investigated the structural determinants responsible for this selectivity of dibromocantharelline using molecular docking (Autodock3.0.5, FlexX and Gold molecular docking packages) and MD simulations (AMBER

Arfeen and Bharatam

software). Hymenialdisine when docked into the active site of GSK-3 and CDK-5 showed uniformity in binding modes. Dibromocantharelline could not be docked into the active site of the CDK-5 by any of the three molecular docking software packages, but showed uniformity in the binding modes when docked into the active site of GSK-3. The reason stated for this difference is the configuration of guanidine ring which is in the same plane as ring B in case of hymenialdisine but is below the plane in case of dibromocantharelline with respect to ring B (Fig. 21). Below the plane orientation of guanidine ring demands for more flat and larger active site, available only in the case of GSK-3. Another reason claimed for the observed difference in the binding mode of dibromocantharelline was the outside deviation of Asp200 side chain from the cavity, creating enough space for the guanidine ring. In case of CDK-5 equivalent residue is Asp144, the side chain of which is directed towards the cavity. The reason again for this is the difference in the orientation of the side chains of residues Phe67, and Glu97 in case of GSK-3 and residues Tyr15, Glu51 and Asp144 in case of CDK-5 which results in firm locking of the Asp144 side chain causing its restricted movement. The weak electrostatic interaction network that exists over Lys85 (in GSK-3) as compared to Lys33 (in CDK-5) renders the side chain of Asp200 to move and allows the accommodation of larger groups. To validate the above mentioned theory an MD simulation study was performed and it was found that during the simulation run, the side chain of Asp200 indeed moves away from the cavity allowing the better accommodation of guanidine ring. The docking study of dibromocantharelline also showed all the important hydrogen bond interactions, which included (i) hydrogen bond interaction between N atom (Ring A) with the carbonyl oxygen of Val135, (ii) salt bridge involving the O atom of the B ring and the side chain of Arg141 and an (iii) electrostatic interaction involving residues Gln185, Asn186 and guanidine ring. Using the above discussed molecular dynamic simulation studies; the key interactions that govern the stability of the substrate bound the GSK-3ß complexity and importance of the catalytic triad in GSK-3 have been identified. The inhibition by binding at the substrate binding site is the future of GSK-3 drug discovery; in this context the above mentioned studies have provided important clues which can play a significant role while designing the new inhibitors. Lu et al. through ONIOM and molecular dynamic simulation studies proven that conserved water molecule stabilizes the inhibitor-enzyme complex. Therefore the water molecules involved in bridged hydrogen bonding should be retained in the active site while performing the molecular docking and MD simulation studies on ATP competitive inhibitors. The MD simulation studies have also been carried to explore the possible reasons of selectivity for certain ATP competitive inhibitors but the number of reports are very less. In future more efforts based on MD simulation studies, especially addressing the lead optimization issue, are required.

Fig. (20). Structure of different paullones 1-azakenpaullone, 2-azakenpaullone, alsterpaullone.



17

Fig. (21). Structures of hymenialdisine and dibromocantharelline.

8. CONCLUSIONS AND FUTURE PROJECTIONS The role of GSK-3 in Type-II diabetes and Alzheimer’s pathology is well established, but its role in cancer is controversial and still needs to be proven, though definite evidences in some form of cancer have been published. The lack of selectivity of ATP competitive inhibitors forced the research groups to look for alternate strategies like (i) blocking substrate binding domain, (ii) blocking catalytic triad domain, (iii) using peptide like molecules which can block both centers, (iv) ligands binding at allosteric site, (v) irreversible modification of Cys199, etc. However there are very few reports for the above mentioned inhibitory mechanism and needs to be extensively explored. 3D QSAR studies have been used to optimize leads like pyrazolo[3,4-b]pyrid[az]ine, pyrazolopyrimidine, 3anilino-4-arylmaleimides, etc. Molecular docking studies have been carried out to predict the binding mode of non-ATP class of GSK-3 inhibitors, pharmacophore mapping along with virtual screening have also been taken up to identify the new leads from the large existing databases. 3D QSAR along with molecular docking, pharmacophore and virtual screening have been attempted to design suitable protocols for the identification of ATP competitive leads that can have the potential of being developed into a drug candidate. All these studies have resulted in the accumulation of a vast amount of knowledge regarding the GSK-3 structure that can be efficiently exploited by the research groups for the design of new GSK-3 inhibitors. The appearance of selective pyrazine derivatives and oxadiazole derivatives with improved selectivity profile are very recent examples of “enhanced knowledge guiding the design of inhibitors”. The four new drugable sites identified by Palomo et al. using fpocket program also needs to be explored further. MD simulation studies have only been taken to address the structure vs. function phenomena associated to GSK-3. This methodology is not being routinely used for the purpose of drug discovery. Clearly there is a wide scope for MD simulation studies to be taken up for the above mentioned purpose. The examples presented in this review points out that there is an opportunity for collective efforts to be taken up, using molecular modeling, organic synthesis and structural biology in designing new inhibitors of GSK-3.

[2] [3] [4] [5]

[6] [7] [8] [9] [10] [11] [12]

[13] [14] [15] [16]

[17] [18]

[19]

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

[20]

ACKNOWLEDGEMENTS Declared none.

[21]

REFERENCES

[22]

[1]

Liao JJ. Molecular Recognition of Protein Kinase Binding Pockets for Design of Potent and Selective Kinase Inhibitors. J Med Chem 2007; 50: 409-24.

Morphy R. Selectively nonselective kinase inhibition: striking the right balance. J Med Chem 2010; 53: 1413-37. Cohen P. Protein kinases--the major drug targets of the twenty-first century? Nat Rev Drug Discov 2002; 1: 309-15. Garcia-Echeverria C, Traxler P, Evans DB. ATP site-directed competitive and irreversible inhibitors of protein kinases. Med Res Rev 2000; 20: 28-57. Noble ME, Endicott JA, Johnson LN. Protein kinase inhibitors: insights into drug design from structure. Science 2004; 303: 18005. Sridhar R, Hanson-Painton O, Cooper DR. Protein kinases as therapeutic targets. Pharm Res 2000; 17: 1345-53. Patel DS, Dessalew N, Iqbal P, Bharatam PV. Structure-based approaches in the design of GSK-3 selective inhibitors. Curr Protein Pept Sci 2007; 8: 352-64. Harwood AJ. Regulation of GSK-3: a cellular multiprocessor. Cell 2001; 105: 821-4. Renfrey S, Downton C, Featherstone J. The painful reality. Nat Rev Drug Discov 2003; 2: 175-6. Frame S, Cohen P. GSK-3 takes centre stage more than 20 years after its discovery. Biochem J 2001; 359: 1-16. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multitasking kinase. J Cell Sci 2003; 116: 1175-86. Van Wauwe J, Haefner B. Glycogen synthase kinase-3 as drug target: from wallflower to center of attention. Drug News Perspect 2003; 16: 557-65. Cohen P, Frame S. The renaissance of GSK-3. Nat Rev Mol Cell Biol 2001; 2: 769-76. Eldar-Finkelman H. Glycogen synthase kinase 3: an emerging therapeutic target. Trends Mol Med 2002; 8: 126-32. Wagman AS, Johnson KW, Bussiere DE. Discovery and development of GSK-3 inhibitors for the treatment of type 2 diabetes. Curr Pharm Des 2004; 10: 1105-37. Ali A, Hoeflich KP, Woodgett JR. Glycogen synthase kinase-3: properties, functions, and regulation. Chem Rev 2001; 101: 252740. Dorronsoro I, Castro A, Martinez A. Inhibitors of glycogen synthase kinase-3: future therapy for unmet medical needs? Expert Opinion on Therapeutic Patents 2002; 12: 1527-36. Kim DJ, Reddy K, Kim MO, et al. (3-Chloroacetyl)-indole, a novel allosteric AKT inhibitor, suppresses colon cancer growth in vitro and in vivo. Cancer Prev Res (Phila) 4: 1842-51. Martinez A, Castro A, Dorronsoro I, Alonso M. Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation. Med Res Rev 2002; 22: 373-84. Martinez A, Gil C, Perez DI. Glycogen synthase kinase 3 inhibitors in the next horizon for Alzheimer's disease treatment. Int J Alzheimers Dis 2011; 2011: 280502. Cohen P, Goedert M. GSK-3 inhibitors: development and therapeutic potential. Nat Rev Drug Discov 2004; 3: 479-87. Grimes CA, Jope RS. The multifaceted roles of glycogen synthase kinase 3 in cellular signaling. Prog Neurobiol 2001; 65: 391-426.

18 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 [23]

[24] [25]

[26] [27]

[28] [29] [30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] [38] [39]

[40] [41] [42]

[43] [44]

[45]

Henriksen EJ, Dokken BB. Role of glycogen synthase kinase-3 in insulin resistance and type 2 diabetes. Curr Drug Targets 2006; 7: 1435-41. Gould TD. Targeting glycogen synthase kinase-3 as an approach to develop novel mood-stabilising medications. Expert Opin Ther Targets 2006; 10: 377-92. Lee J, Kim MS. The role of GSK-3 in glucose homeostasis and the development of insulin resistance. Diabetes Res Clin Pract 2007; 77 Suppl 1: S49-57. Ranatunga S, Del Valle JR. Synthesis of GSK3beta mimetic inhibitors of Akt featuring a novel extended dipeptide surrogate. Bioorg Med Chem Lett 21: 7166-9. Morrow JK, Du-Cuny L, Chen L, et al. Recent development of anticancer therapeutics targeting Akt. Recent Pat Anticancer Drug Discov 6: 146-59. Collins I. Targeted small-molecule inhibitors of protein kinase B as anticancer agents. Anticancer Agents Med Chem 2009; 9: 32-50. McDonnell SR, Hwang SR, Basrur V, et al. NPM-ALK signals through glycogen synthase kinase 3beta to promote oncogenesis. Oncogene 2012; 31: 3733-40. Liu JJ, Dai XJ, Xu Y, et al. Inhibition of lymphoma cell proliferation by peroxisomal proliferator-activated receptor-gamma ligands via Wnt signaling pathway. Cell Biochem Biophys 2012; 62: 1927. Mills CN, Nowsheen S, Bonner JA, Yang ES. Emerging roles of glycogen synthase kinase 3 in the treatment of brain tumors. Front. Mol Neurosci 2012; 4: 47. Mora-Santos M, Limon-Mortes MC, Giraldez S, et al. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors. J Biol Chem 2012; 286: 30047-56. Wang Z, Smith KS, Murphy M, Piloto O, Somervaille TC, Cleary ML. Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy. Nature 2008; 455: 1205-9. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 1980; 107: 519-27. Picton C, Aitken A, Bilham T, Cohen P. Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. Eur J Biochem 1982; 124: 37-45. Woodgett JR, Cohen P. Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase5). Biochim Biophys Acta 1984; 788: 339-47. Kockeritz L, Doble B, Patel S, Woodgett JR. Glycogen synthase kinase-3--an overview of an over-achieving protein kinase. Curr Drug Targets 2006; 7: 1377-88. Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 1990; 9: 2431-8. Tsai LH, Harlow E, Meyerson M. Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase. Nature 1991; 353: 174-7. Meyerson M, Enders GH, Wu CL, et al. A family of human cdc2related protein kinases. EMBO J 1992; 11: 2909-17. Hardcastle IR, Golding BT, Griffin RJ. Designing inhibitors of cyclin-dependent kinases. Annu Rev Pharmacol Toxicol 2002; 42: 325-48. Bain J, McLauchlan H, Elliott M, Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J 2003; 371: 199204. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351: 95-105. Lawrie AM, Noble ME, Tunnah P, Brown NR, Johnson LN, Endicott JA. Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2. Nat Struct Biol 1997; 4: 796-801. Cline GW, Johnson K, Regittnig W, et al. Effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in Zucker diabetic fatty (fa/fa) rats. Diabetes 2002; 51: 2903-10.

Arfeen and Bharatam [46]

[47]

[48]

[49] [50]

[51] [52]

[53]

[54] [55]

[56] [57]

[58]

[59] [60]

[61]

[62] [63]

[64] [65] [66] [67]

[68] [69]

Meijer L, Thunnissen AM, White AW, et al. Inhibition of cyclindependent kinases, GSK-3 and CK1 by hymenialdisine, a marine sponge constituent. Chem Biol 2000; 7: 51-63. Stukenbrock H, Mussmann R, Geese M, et al. 9-cyano-1azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication. J Med Chem 2008; 51: 2196-207. Witherington J, Bordas V, Gaiba A, et al. 6-heteroarylpyrazolo[3,4-b]pyridines: Potent and Selective Inhibitors of Glycogen Synthase Kinase-3 (GSK-3). Bioorg Med Chem Lett 2003; 13: 3059-62. Smith DG, Buffet M, Fenwick AE, et al. 3-Anilino-4arylmaleimides: potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3). Bioorg Med Chem Lett 2001; 11: 635-9. Toledo LM, Lydon NB. Structures of staurosporine bound to CDK2 and cAPK--new tools for structure-based design of protein kinase inhibitors. Structure 1997; 5: 1551-6. Zhang HC, Ye H, Conway BR, et al. 3-(7-Azaindolyl)-4arylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3. Bioorg Med Chem Lett 2004; 14: 3245-50. Olesen PH, Sorensen AR, Urso B, et al. Synthesis and in vitro characterization of 1-(4-aminofurazan-3-yl)-5-dialkylaminomethyl1H-[1,2,3]triazole-4-carboxyl ic acid derivatives. A new class of selective GSK-3 inhibitors. J Med Chem 2003; 46: 3333-41. Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins. Chem Biol 2003; 10: 1255-66. Kunick C, Lauenroth K, Leost M, Meijer L, Lemcke T. 1Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3. Bioorg Med Chem Lett 2004; 14: 413-6. Leost M, Schultz C, Link A, et al. Paullones are potent inhibitors of glycogen synthase kinase-3 and cyclin-dependent kinase 5/p25. Eur J Biochem 2000; 267: 5983-94. Bartlett S, Beddard GS, Jackson RM, et al. Comparison of the ATP binding sites of protein kinases using conformationally diverse bisindolylmaleimides. J Am Chem Soc 2005; 127: 11699-708. Vulpetti A, Crivori P, Cameron A, et al. Structure-based approaches to improve selectivity: CDK2-GSK-3 binding site analysis. J Chem Inf Model 2005; 45: 1282-90. Polychronopoulos P, Magiatis P, Skaltsounis AL, et al. Structural Basis for the Synthesis of Indirubins as Potent and Selective Inhibitors of Glycogen Synthase Kinase-3 and Cyclin-Dependent Kinases. J Med Chem 2004; 47: 935-46. Dessalew N, Bharatam PV. Identification of potential glycogen kinase-3 inhibitors by structure based virtual screening. Biophys Chem 2007; 128: 165-75. Kozikowski AP, Gaisina IN, Yuan H, et al. Structure-based design leads to the identification of lithium mimetics that block mania-like effects in rodents. possible new GSK-3 therapies for bipolar disorders. J Am Chem Soc 2007; 129: 8328-32. Patel DS, Bharatam PV. New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies. J Comput Aided Mol Des 2006; 20: 55-66. Vadivelan S, Sinha BN, Tajne S, Jagarlapudi SA. Fragment and knowledge-based design of selective GSK-3 inhibitors using virtual screening models. Eur J Med Chem 2009; 44: 2361-71. Gao C, Holscher C, Liu Y, Li L. GSK3: a key target for the development of novel treatments for type 2 diabetes mellitus and Alzheimer disease. Rev Neurosci 2011; 23: 1-11. Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: more than a namesake. Br J Pharmacol 2009; 156: 885-98. Luo J. Glycogen synthase kinase 3beta (GSK3beta) in tumorigenesis and cancer chemotherapy. Cancer Lett 2009; 273: 194-200. Ougolkov AV, Billadeau DD. Targeting GSK-3: a promising approach for cancer therapy? Future Oncol 2006; 2: 91-100. Meijer L, Flajolet M, Greengard P. Pharmacological inhibitors of glycogen synthase kinase 3. Trends Pharmacol Sci 2004; 25: 47180. Eldar-Finkelman H, Martinez A. GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS. Front Mol Neurosci 2011; 4: 32. Plyte SE, Feoktistova A, Burke JD, Woodgett JR, Gould KL. Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant. Mol Cell Biol 1996; 16: 179-91.

Design of GSK-3 Inhibitors: An Overview on Recent Advancements [70]

[71] [72]

[73] [74]

[75]

[76] [77]

[78] [79]

[80] [81]

[82] [83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

Plyte SE, Feoktistova A, Burke JD, Woodgett JR, Gould KL. Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant. Mol Cell Biol 1997; 17: 1756. Woodgett JR. cDNA cloning and properties of glycogen synthase kinase-3. Methods Enzymol 1991; 200: 564-77. Shaw PC, Davies AF, Lau KF, et al. Isolation and chromosomal mapping of human glycogen synthase kinase-3 and -3 encoding genes. Genome 1998; 41: 720-7. Wehrli M, Dougan ST, Caldwell K, et al. arrow encodes an LDLreceptor-related protein essential for Wingless signalling. Nature 2000; 407: 527-30. Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. Requirement for glycogen synthase kinase-3 in cell survival and NF-kappaB activation. Nature 2000; 406: 86-90. Ruel L, Bourouis M, Heitzler P, Pantesco V, Simpson P. Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of Notch. Nature 1993; 362: 557-60. Lee HC, Tsai JN, Liao PY, et al. Glycogen synthase kinase 3 and 3 have distinct functions during cardiogenesis of zebrafish embryo. BMC. Dev. Biol 2007; 7: 93. Markou T, Cullingford TE, Giraldo A, et al. Glycogen synthase kinases 3 and 3 in cardiac myocytes: regulation and consequences of their inhibition. Cell Signal 2008; 20: 206-18. Mukai F, Ishiguro K, Sano Y, Fujita SC. Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3. J Neurochem 2002; 81: 1073-83. ter Haar E, Coll JT, Austen DA, Hsiao HM, Swenson L, Jain J. Structure of GSK-3 reveals a primed phosphorylation mechanism. Nat Struct. Biol 2001; 8: 593-6. Dajani R, Fraser E, Roe SM, et al. Crystal structure of glycogen synthase kinase 3: structural basis for phosphate-primed substrate specificity and autoinhibition. Cell 2001; 105: 721-32. Pearl LH, Barford D. Regulation of protein kinases in insulin, growth factor and Wnt signalling. Curr. Opin. Struct. Biol 2002; 12: 761-7. Arnost M, Pierce A, ter Haar E, et al. 3-Aryl-4-(arylhydrazono)1H-pyrazol-5-ones: Highly ligand efficient and potent inhibitors of GSK-3. Bioorg. Med Chem Lett 2010; 20: 1661-4. Bertrand JA, Thieffine S, Vulpetti A, et al. Structural characterization of the GSK-3 active site using selective and non-selective ATP-mimetic inhibitors. J Mol Biol 2003; 333: 393-407. Moreno FJ, Munoz-Montano JR, Avila J. Glycogen synthase kinase 3 phosphorylation of different residues in the presence of different factors: analysis on tau protein. Mol Cell Biochem 1996; 165: 47-54. Ilouz R, Kowalsman N, Eisenstein M, Eldar-Finkelman H. Identification of novel glycogen synthase kinase-3 substrate-interacting residues suggests a common mechanism for substrate recognition. J Biol Chem 2006; 281: 30621-30. Lu SY, Jiang YJ, Zou JW, Wu TX. Molecular Modeling and Molecular Dynamics Simulation Studies of the GSK-3/ATP/Substrate Complex: Understanding the Unique P+4 Primed Phosphorylation Specificity for GSK-3 Substrates. J Chem Inf. Model 2011; DOI: 10.1021/ci100493j. Frame S, Cohen P, Biondi RM. A common phosphate binding site explains the unique substrate specificity of GSK-3 and its inactivation by phosphorylation. Mol Cell 2001; 7: 1321-7. Fiol CJ, Mahrenholz AM, Wang Y, Roeske RW, Roach PJ. 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 1987; 262: 14042-8. Hughes K, Ramakrishna S, Benjamin WB, Woodgett JR. Identification of multifunctional ATP-citrate lyase kinase as the alphaisoform of glycogen synthase kinase-3. Biochem J 1992; 288 ( Pt 1): 309-14. Benjamin WB, Pentyala SN, Woodgett JR, Hod Y, Marshak D. ATP citrate-lyase and glycogen synthase kinase-3 in 3T3-L1 cells during differentiation into adipocytes. Biochem J 1994; 300 ( Pt 2): 477-82. Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S, Kikuchi A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3 and beta-catenin and promotes GSK-3-


[92]

[93]

[94]

[95]

[96] [97]

[98] [99]

[100] [101]

[102] [103]

[104]

[105] [106] [107]

[108]

[109]

[110] [111]

[112] [113]

19

dependent phosphorylation of -catenin. EMBO J 1998; 17: 137184. Pulverer BJ, Fisher C, Vousden K, Littlewood T, Evan G, Woodgett JR. Site-specific modulation of c-Myc cotransformation by residues phosphorylated in vivo. Oncogene 1994; 9: 59-70. Hanger DP, Hughes K, Woodgett JR, Brion JP, Anderton BH. Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci. Lett 1992; 147: 58-62. Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT. The axis-inducing activity, stability, and subcellular distribution of -catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 1996; 10: 1443-54. Bengtsson E. The aqueous flare inducing effect of prostaglandin and alpha-melanocyte stimulating hormone [proceedings]. Acta. Ophthalmol. Suppl 1975: 55-7. Frame S, Zheleva D. Targeting glycogen synthase kinase-3 in insulin signalling. Expert Opin. Ther. Targets 2006; 10: 429-44. Thomas GM, Frame S, Goedert M, Nathke I, Polakis P, Cohen P. A GSK-3 binding peptide from FRAT1 selectively inhibits the GSK3catalysed phosphorylation of axin and -catenin. FEBS Lett 1999; 458: 247-51. Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci 2002; 27: 514-20. Brown NR, Noble ME, Endicott JA, Johnson LN. The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat Cell. Biol 1999; 1: 438-43. Keskin O. Comparison of full-atomic and coarse-grained models to examine the molecular fluctuations of c-AMP dependent protein kinase. J Biomol. Struct. Dyn 2002; 20: 333-45. Kaidanovich-Beilin O, Eldar-Finkelman H. Peptides targeting protein kinases: strategies and implications. Physiology (Bethesda) 2006; 21: 411-8. Eldar-Finkelman H, Eisenstein M. Peptide inhibitors targeting protein kinases. Current pharmaceutical design 2009; 15: 24632470. Gartner A, Huang X, Hall A. Neuronal polarity is regulated by glycogen synthase kinase-3 (GSK-3) independently of Akt/PKB serine phosphorylation. J Cell Sci 2006; 119: 3927-34. Wang QM, Fiol CJ, DePaoli-Roach AA, Roach PJ. Glycogen synthase kinase-3 is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem 1994; 269: 14566-74. Echeverria-Coello CR. [Milk production in the nutrition situation in Mexico]. Gac. Med Mex 1975; 110: 13-5. McManus EJ, Sakamoto K, Armit LJ, et al. Role that phosphorylation of GSK-3 plays in insulin and Wnt signalling defined by knockin analysis. EMBO J 2005; 24: 1571-83. Nikoulina SE, Ciaraldi TP, Mudaliar S, Mohideen P, Carter L, Henry RR. Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes. Diabetes 2000; 49: 263-71. Nikoulina SE, Ciaraldi TP, Mudaliar S, Carter L, Johnson K, Henry RR. Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle. Diabetes 2002; 51: 2190-8. Ciaraldi TP, Nikoulina SE, Henry RR. Role of glycogen synthase kinase-3 in skeletal muscle insulin resistance in Type 2 diabetes. J Diabetes. Complications 2002; 16: 69-71. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995; 378: 785-9. Welsh GI, Miller CM, Loughlin AJ, Price NT, Proud CG. Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase-3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin. FEBS Lett 1998; 421: 125-30. Thornton TM, Pedraza-Alva G, Deng B, et al. Phosphorylation by p38 MAPK as an alternative pathway for GSK-3 inactivation. Science 2008; 320: 667-70. Martinez A, Alonso M, Castro A, et al. SAR and 3D-QSAR Studies on Thiadiazolidinone Derivatives: Exploration of Structural Requirements for Glycogen Synthase Kinase 3 Inhibitors. J Med Chem 2005; 48: 7103-12.

20 Current Pharmaceutical Design, 2013, Vol. 19, No. 00 [114]

[115] [116]

[117]

[118] [119]

[120]

[121]

[122] [123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131] [132]

[133]

Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS. Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J Biol Chem 2003; 278: 33067-77. Berridge MJ, Downes CP, Hanley MR. Neural and developmental actions of lithium: a unifying hypothesis. Cell 1989; 59: 411-9. Gould TD, Manji HK. Glycogen synthase kinase-3: a putative molecular target for lithium mimetic drugs. Neuropsychopharmacology 2005; 30: 1223-37. Ilouz R, Kaidanovich O, Gurwitz D, Eldar-Finkelman H. Inhibition of glycogen synthase kinase-3 by bivalent zinc ions: insight into the insulin-mimetic action of zinc. Biochem Biophys. Res Commun 2002; 295: 102-6. Atilla-Gokcumen GE, Pagano N, Streu C, et al. Extremely tight binding of a ruthenium complex to glycogen synthase kinase 3. Chembiochem 2008; 9: 2933-6. Bregman H, Carroll PJ, Meggers E. Rapid access to unexplored chemical space by ligand scanning around a ruthenium center: discovery of potent and selective protein kinase inhibitors. J Am Chem Soc 2006; 128: 877-84. Saitoh M, Kunitomo J, Kimura E, et al. Design, synthesis and structure-activity relationships of 1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3. Bioorg. Med Chem Lett 2009; 17: 2017-29. Saitoh M, Kunitomo J, Kimura E, et al. 2-{3-[4(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4oxadiazol e derivatives as novel inhibitors of glycogen synthase kinase-3 with good brain permeability. J Med Chem 2009; 52: 6270-86. Lo Monte F, Kramer T, Gu J, et al. Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3alpha. J Med Chem 2012; 55: 4407-24. Zou H, Zhou L, Li Y, et al. Benzo[e]isoindole-1,3-diones as potential inhibitors of glycogen synthase kinase-3 (GSK-3). Synthesis, kinase inhibitory activity, zebrafish phenotype, and modeling of binding mode. J Med Chem 2010; 53: 994-1003. Khanfar MA, Asal BA, Mudit M, Kaddoumi A, El Sayed KA. The marine natural-derived inhibitors of glycogen synthase kinase-3 phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling. Bioorg. Med Chem 2009; 17: 60326039. Khanfar MA, Hill RA, Kaddoumi A, El Sayed KA. Discovery of novel GSK-3 inhibitors with potent in vitro and in vivo activities and excellent brain permeability using combined ligand- and structure-based virtual screening. J Med Chem 2010; 53: 8534-45. Rochais C, Duc NV, Lescot E, et al. Synthesis of new dipyrroloand furopyrrolopyrazinones related to tripentones and their biological evaluation as potential kinases (CDKs1-5, GSK-3) inhibitors. Eur. J Med Chem 2009; 44: 708-16. Berg S, Bergh M, Hellberg S, et al. Discovery of Novel Potent and Highly Selective Glycogen Synthase Kinase-3beta (GSK3beta) Inhibitors for Alzheimer's Disease: Design, Synthesis, and Characterization of Pyrazines. J Med Chem 2012; DOI: 10.1021/jm201724m. Martinez A, Alonso M, Castro A, Perez C, Moreno FJ. First nonATP competitive glycogen synthase kinase 3 (GSK-3) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer's disease. J Med Chem 2002; 45: 1292-9. Castro A, Encinas A, Gil C, et al. Non-ATP Competitive Glycogen Synthase Kinase 3 (GSK-3) Inhibitors: Study of Structural Requirements for Thiadiazolidinone derivatives. Bioorg. Med Chem 2008; 16: 495-510. Palomo V, Perez DI, Perez C, et al. 5-Imino-1,2,4-Thiadiazoles: First Small Molecules as Substrate Competitive Inhibitors of Glycogen Synthase Kinase 3. J Med Chem 2012. Mazanetz MP, Fischer PM. Untangling tau Hyperphosphorylation in Drug Design for Neurodegenerative Diseases. Nat Rev Drug Discov 2007; 6: 464-79. Hamann M, Alonso D, Martin-Aparicio E, et al. Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure-activity relationship (SAR) studies of the manzamine alkaloids. Potential for Alzheimer's disease. J Nat Prod 2007; 70: 1397-405. Rao KV, Kasanah N, Wahyuono S, Tekwani BL, Schinazi RF, Hamann MT. Three new manzamine alkaloids from a common Indonesian sponge and their activity against infectious and tropical parasitic diseases. J Nat Prod 2004; 67: 1314-8.

Arfeen and Bharatam [134]

[135]

[136] [137]

[138]

[139]

[140]

[141] [142]

[143]

[144] [145]

[146] [147]

[148]

[149] [150]

[151]

[152] [153] [154] [155]

Rao KV, Donia MS, Peng J, et al. Manzamine B and E and Ircinal A Related Alkaloids from an Indonesian Acanthostrongylophora Sponge and their Activity against Infectious, tropical parasitic, and Alzheimer's diseases. J Nat Prod 2006; 69: 1034-40. Yousaf M, Hammond NL, Peng J, et al. New manzamine alkaloids from an Indo-Pacific sponge. Pharmacokinetics, oral availability, and the significant activity of several manzamines against HIV-I, AIDS opportunistic infections, and inflammatory diseases. J Med Chem 2004; 47: 3512-7. Laport MS, Santos OC, Muricy G. Marine sponges: potential sources of new antimicrobial drugs. Curr. Pharm. Biotechnol 2009; 10: 86-105. Rao KV, Santarsiero BD, Mesecar AD, Schinazi RF, Tekwani BL, Hamann MT. New manzamine alkaloids with activity against infectious and tropical parasitic diseases from an Indonesian sponge. J Nat Prod 2003; 66: 823-8. Peng J, Kudrimoti S, Prasanna S, et al. Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections. J Med Chem 2010; 53: 61-76. Conde S, Perez DI, Martinez A, Perez C, Moreno FJ. Thienyl and phenyl alpha-halomethyl ketones: new inhibitors of glycogen synthase kinase (GSK-3) from a library of compound searching. J Med Chem 2003; 46: 4631-3. Perez DI, Conde S, Perez C, et al. Thienylhalomethylketones: Irreversible glycogen synthase kinase 3 inhibitors as useful pharmacological tools. Bioorg. Med Chem 2009; 17: 6914-25. Perez DI, Palomo V, Perez C, et al. Switching reversibility to irreversibility in glycogen synthase kinase 3 inhibitors: clues for specific design of new compounds. J Med Chem 2011; 54: 4042-56. Bax B, Carter PS, Lewis C, et al. The structure of phosphorylated GSK-3 complexed with a peptide, FRATtide, that inhibits betacatenin phosphorylation. Structure 2001; 9: 1143-52. Plotkin B, Kaidanovich O, Talior I, Eldar-Finkelman H. Insulin mimetic action of synthetic phosphorylated peptide inhibitors of glycogen synthase kinase-3. J Pharmacol. Exp. Ther 2003; 305: 974-80. Palomo V, Soteras I, Perez DI, et al. Exploring the binding sites of glycogen synthase kinase 3. Identification and characterization of allosteric modulation cavities. J Med Chem 2011; 54: 8461-70. Eldar-Finkelman H, Licht-Murava A, Pietrokovski S, Eisenstein M. Substrate competitive GSK-3 inhibitors - strategy and implications. Biochim. Biophys. Acta 2010; 1804: 598-603. Bhat R, Xue Y, Berg S, et al. Structural Insights and Biological Effects of Glycogen Synthase Kinase 3 Specific Inhibitor ARA014418. J Biol Chem 2003; 278: 45937-45. Ring DB, Johnson KW, Henriksen EJ, et al. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes 2003; 52: 588-95. Coghlan MP, Culbert AA, Cross DA, et al. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol 2000; 7: 793-803. Fischer PM. CDK versus GSK-3 inhibition: a purple haze no longer? Chem Biol 2003; 10: 1144-6. Kozikowski AP, Gaisina IN, Petukhov PA, et al. Highly Potent and Specific GSK-3 inhibitors That Block tau Phosphorylation and Decrease alpha-Synuclein Protein Expression in a Cellular Model of Parkinson's disease. ChemMedChem 2006; 1: 256-66. Caballero J, Zilocchi S, Tiznado W, Collina S, Rossi D. Binding studies and quantitative structure-activity relationship of 3-amino1H-indazoles as inhibitors of GSK-3. Chem Biol Drug Des 2011; 78: 631-41. Akhtar M, Bharatam PV. 3D-QSAR and Molecular Docking Studies on 3-Anilino-4-Arylmaleimide Derivatives as Glycogen Synthase Kinase-3 Inhibitors. Chem Biol Drug Des 2011. Garcia I, Fall Y, Gomez G. QSAR, docking, and CoMFA studies of GSK-3 inhibitors. Curr. Pharm. Des 2010; 16: 2666-75. Garcia I, Fall Y, Gomez G. Update of QSAR Docking Studies of the GSK-3 Inhibitors. Current Bioinformatics 2011; 6: 215-232. Kim KH, Gaisina I, Gallier F, et al. Use of molecular modeling, docking, and 3D-QSAR studies for the determination of the binding mode of benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3 inhibitors. J Mol Model 2009; 15: 1463-79.

Design of GSK-3 Inhibitors: An Overview on Recent Advancements [156]

[157]

[158]

[159] [160]

[161] [162]

[163] [164]

[165] [166]

[167] [168] [169] [170] [171]

[172]

[173]

Kunick C, Lauenroth K, Wieking K, et al. Evaluation and comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibition by paullones. J Med Chem 2004; 47: 22-36. Dessalew N, Patel DS, Bharatam PV. 3D-QSAR and molecular docking studies on pyrazolopyrimidine derivatives as glycogen synthase kinase-3 inhibitors. J Mol Graph. Model 2007; 25: 88595. Lescot E, Bureau R, Sopkova-de Oliveira Santos J, et al. 3D-QSAR and docking studies of selective GSK-3 inhibitors. Comparison with a thieno[2,3-b]pyrrolizinone derivative, a new potential lead for GSK-3 ligands. J Chem Inf. Model 2005; 45: 708-15. Patel DS, Bharatam PV. Selectivity criterion for pyrazolo[3,4b]pyrid[az]ine derivatives as GSK-3 inhibitors: CoMFA and molecular docking studies. Eur. J Med Chem 2008; 43: 949-57. Taha MO, Bustanji Y, Al-Ghussein MA, et al. Pharmacophore modeling, quantitative structure-activity relationship analysis, and in silico screening reveal potent glycogen synthase kinase-3 inhibitory activities for cimetidine, hydroxychloroquine, and gemifloxacin. J Med Chem 2008; 51: 2062-77. Fang J, Huang D, Zhao W, Ge H, Luo HB, Xu J. A new protocol for predicting novel GSK-3 ATP competitive inhibitors. J Chem Inf. Model 2010; 51: 1431-8. Bharatam PV, Khanna S, Francis SM. Modelling and informatics in drug design. In: Shyane CG, ed.^eds., Preclinical Development Handbook. Wiley 2008; pp. 1-45. Liu L, Zhang L-N, Jiang F-C. Construction of the Pharmacophore Model of Glycogen Synthase Kinase-3 Inhibitors. Chinese Journal of Chemistry 2007; 25: 892-897. Dessalew N, Bharatam PV. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening. Chem Biol Drug Des 2006; 68: 154-65. Kim HJ, Choo H, Cho YS, No KT, Pae AN. Novel GSK-3 inhibitors from sequential virtual screening. Bioorg. Med Chem 2008; 16: 636-43. Polgar T, Baki A, Szendrei GI, Keseru GM. Comparative virtual and experimental high-throughput screening for glycogen synthase kinase-3 inhibitors. J Med Chem 2005; 48: 7946-59. Eglen RM, Reisine T. The current status of drug discovery against the human kinome. Assay Drug Dev. Technol 2009; 7: 22-43. Eglen R, Reisine T. Drug discovery and the human kinome: recent trends. Pharmacol. Ther 2011; 130: 144-56. McInnes C, Fischer PM. Strategies for the design of potent and selective kinase inhibitors. Curr. Pharm. Des 2005; 11: 1845-63. Bogoyevitch MA, Fairlie DP. A new paradigm for protein kinase inhibition: blocking phosphorylation without directly targeting ATP binding. Drug Discov. Today 2007; 12: 622-33. Lu SY, Jiang YJ, Lv J, Zou JW, Wu TX. Role of bridging water molecules in GSK-3-inhibitor complexes: insights from QM/MM, MD, and molecular docking studies. J Comput. Chem 2011; 32: 1907-18. Buch I, Fishelovitch D, London N, Raveh B, Wolfson HJ, Nussinov R. Allosteric regulation of glycogen synthase kinase 3: a theoretical study. Biochemistry 2011; 49: 10890-901. Chen Q, Cui W, Cheng Y, Zhang F, Ji M. Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dy-

Received: October 22, 2012

Accepted: December 17, 2012


[174]

[175]

[176]

[177]

[178] [179] [180]

[181]

[182] [183]

[184]

[185] [186]

[187] [188] [189] [190]

[191]

21

namics simulations and free-energy calculations. J Mol Model 2011; 17: 795-803. Sun H, Jiang YJ, Yu QS, Luo CC, Zou JW. Effect of mutation K85R on GSK-3: Molecular dynamics simulation. Biochem Biophys. Res Commun 2008; 377: 962-5. Zhang N, Zhong R, Yan H, Jiang Y. Structural features Underlying Selective Inhibition of GSK-3 by Dibromocantharelline: Implications for Rational Drug Design. Chem Biol Drug Des 2011; 77: 199-205. Bhat RV, Shanley J, Correll MP, et al. Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3 in cellular and animal models of neuronal degeneration. Proc. Natl. Acad. Sci. U S A 2000; 97: 11074-9. Lucas JJ, Hernandez F, Gomez-Ramos P, Moran MA, Hen R, Avila J. Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3 conditional transgenic mice. EMBO J 2001; 20: 27-39. Dajani R, Fraser E, Roe SM, et al. Structural basis for recruitment of glycogen synthase kinase 3 to the axin-APC scaffold complex. EMBO J 2003; 22: 494-501. Allard J, Nikolcheva T, Gong L, et al. To be published www.rcsb.org/pdb, PDB code1R0E. Shin D, Lee SC, Heo YS, et al. Design and synthesis of 7-hydroxy1H-benzoimidazole derivatives as novel inhibitors of glycogen synthase kinase-3. Bioorg. Med Chem Lett 2007; 17: 5686-9. Zhang HC, Bonaga LV, Ye H, Derian CK, Damiano BP, Maryanoff BE. Novel bis(indolyl)maleimide pyridinophanes that are potent, selective inhibitors of glycogen synthase kinase-3. Bioorg. Med Chem Lett 2007; 17: 2863-8. Menichincheri M, Bargiotti A, Berthelsen J, et al. First Cdc7 kinase inhibitors: pyrrolopyridinones as potent and orally active antitumor agents. 2. Lead discovery. J Med Chem 2009; 52: 293-307. Aronov AM, Tang Q, Martinez-Botella G, et al. Structure-guided design of potent and selective pyrimidylpyrrole inhibitors of extracellular signal-regulated kinase (ERK) using conformational control. J Med Chem 2009; 52: 6362-8. Feng L, Geisselbrecht Y, Blanck S, et al. Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors. J Am Chem Soc 2011; 133: 5976-86. Atilla-Gokcumen GE, Di Costanzo L, Meggers E. Structure of anticancer ruthenium half-sandwich complex bound to glycogen synthase kinase 3. J Biol Inorg. Chem 2010; 16: 45-50. Coffman K, Brodney M, Cook J, et al. 6-amino-4-(pyrimidin-4yl)pyridones: novel glycogen synthase kinase-3 inhibitors. Bioorg. Med Chem Lett 2011; 21: 1429-33. Gentile G, Bernasconi G, Pozzan A, et al. Identification of 2-(4pyridyl)thienopyridinones as GSK-3beta inhibitors. Bioorg Med Chem Lett; 21: 4823-7. Mesecar AM, Walters RL. To be published www.rcsb.org/pdb, PDB code 3SD0. Kim HT, Lee SC, Chang HJ. To be published www.rcsb.org/pdb, PDB code 4DIT. Gentile G, Merlo G, Pozzan A, et al. 5-Aryl-4-carboxamide-1,3oxazoles: potent and selective GSK-3 inhibitors. Bioorg Med Chem Lett; 22: 1989-94. Cheng RKY, Rowan F, Barker jj. To be published www.rcsb.org/pdb, PDB code 3SAY.