Current Pharmaceutical Design, 2004, 10, 1105-1137 1105 Discovery and Development of GSK3 Inhibitors for the Treatment of Type 2 Diabetes Allan S. Wagman†,*, Kirk W. Johnson‡ and Dirksen E. Bussiere¥ † Medicinal Chemistry, Chiron Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA 94608-2916, ‡Pharmacology and Preclinical Development, Genesoft Pharmaceuticals, Inc., 7300 Shoreline Ct., South San Francisco, CA 94080, ¥ Computational Chemistry and Structural Biology, Chiron Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA 94608-2916 Abstract: Originally identified as a modulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now understood to play an important regulatory role in a variety of pathways including initiation of protein synthesis, cell proliferation, cell differentiation, apoptosis, and is essential for embryonic development as a component of the Wnt signaling cascade. GSK3 can be considered as a target for both metabolic and neurological disorders. GSK3's association with neuronal apoptosis and hyper-phosphorylation of tau make this kinase an attractive therapeutic target for neurodegenerative conditions such as head trauma, stroke and Alzheimer's disease. While noting GSK3's many associated functions, this review will focus on GSK3 as a central negative regulator in the insulin signaling pathway, its role in insulin resistance, and the utility of GSK3 inhibitors for intervention and control of metabolic diseases including type 2 diabetes. Recent crystal structures, including the active (phosphorylated Tyr-216) form of GSK3β, provide a wealth of structural information and greater understanding of GSK3's unique regulation and substrate specificity. Many potent and selective small molecule inhibitors of GSK3 have now been identified, and used in vitro to modulate glycogen metabolism and gene transcription, increase glycogen synthase activity and enhance insulin-stimulated glucose transport. The pharmacology of potent and selective GSK3 inhibitors (CT 99021 and CT 20026) is described in a number of in vitro and in vivo models following acute or chronic exposure. The efficacy of clinical candidates in diabetic primates and the implications for clinical development are discussed. The profile of activity is consistent with a unique form of insulin sensitization which is well suited for indications such as metabolic syndrome X and type 2 diabetes. INTRODUCTION In recent years there has been a dramatic increase in the global prevalence of diabetes. Incidence is steadily increasing, creating a new burden on health care systems and a huge unmet need for new methods of treatment . The central role of GSK3 in glucose metabolism makes it an exciting target for controlling hyperglycemia, and inhibition of GSK3 activity may represent a novel mechanism for improving glucose disposal in an insulin-conserving manner. GSK3 is one of many signaling components downstream from the insulin receptor (IR), but this kinase has several unique features that make it an attractive target for drug discovery . Unlike other known intracellular protein kinases, GSK3 is constitutively active in resting cells and is inhibited through the action of extracellular signals, such as insulin, and activation of cell signaling pathways. Insulin modulates glycogen accumulation in sensitive tissues by increasing glucose transport and increasing glycogen synthesis. In diabetics, both of these pathways may be defective resulting in diminished glycogen storage. Through the action of insulin, glycogen synthase (GS) is dephosphorylated and activated by an increase in protein phosphatase 1G (PP1G) activity and the inhibition of kinases such as PKA and GSK3 . In response to insulin, IRS-1 *Address correspondence to this author at the Medicinal Chemistry, Chiron Corporation, 4560 Horton Street, M/S 4.5, Emeryville, CA 94608-2916; Tel: (510) 923-7796; Fax: (510) 923-3360; E-mail: [email protected] 1381-6128/04 $45.00+.00 activates akt/PKB which in turn inhibits GSK3. The negative regulation of GSK3 by insulin occurs in the key cell types important for glycogen metabolism: hepatocytes, myocytes, and adipocytes [4-6]. Disregulation or overexpression of GSK3 in these cells could lead to insulin resistance. Thus, inhibiting GSK3 in concert with insulin-induced signaling should increase the activity of GS and improve glycogen deposition in critical glucose-controlling tissues. GSK3 is a cytosolic serine/threonine protein kinase found in two closely related isoforms, GSK3α and GSK3β, which are expressed ubiquitously in mammalian tissues. Both isoforms have nearly identical biochemical functions and substrate affinities , and have been recently reviewed in detail [8-10]. The genes of GSK3α and GSK3 β show a high degree of sequence identity (~85%), while the active sites share 93% identity . GSK3 can be considered an essential gene in that mouse GSK3β knockouts exhibit normal development in utero until liver failure leads to death between E13.5 and E14.5. The lethal phenotype suggested that the GSK3α isoform can not fully compensate for GSK3β deficiency and would indicate certain independent regulatory roles for each isoform . Some differences in gene expression and protein concentration have been found in mammalian tissues that might represent differential regulation, but no strong correlations have emerged . Thus, while some differential expression and effects of the two isozymes have been reported, clear discrimination of functional roles has not been apparent. © 2004 Bentham Science Publishers Ltd. 1106 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Abnormal overexpression of GSK3 in peripheral tissues could potentially contribute to the pathology of diabetes. Studies in vitro show that hyperactive mutants of GSK3 expressed in cells lead to a reduction in GS activity [13, 14]. Compounds known to attenuate insulin signaling, such as okadaic acid and TNFα, increase the serine/threonine phosphorylation of IRS-1 [15, 16]. GSK3 also phosphorylates IRS-1 on serine residues and likewise inhibits insulin signaling in cells . Thus, if serine phosphorylation of IRS-1 contributes to insulin resistance, abnormally high amounts of GSK3 in tissue might lead to impaired insulin signaling and GSK3 would be further enhanced by the impairment signaling through IRS-1. Recently, two studies indicate a potential mechanism by which GSK3 may have a direct effect on glucose transport. In the first study, GSK3 was shown to phosphorylate and inactivate kinesin light chains . The second study links kinesin to the regulation of membrane trafficking of glucose transport protein-4 (GLUT4) vesicles to the plasma membrane . Overall, GSK3 has the potential to interfere with normal insulin signaling through hyperactivity or overexpression in muscle, adipose or liver. Insulin resistance could arise through GSK3-dependent phosphorylation of GS, IRS-1 or kinesins involved in GLUT-4 translocation. To further define GSK3's role in insulin resistance and other pathways, highly potent, selective and cellpermeable GSK3 inhibitors have been useful biochemical probes. Compounds such as CT 99021 and CT 20026 have provided a roadmap for better understanding the role of GSK3 in glucose metabolism and the potential of GSK3 inhibitors as metabolic disease therapeutics. BIOLOGY, ENZYMOLOGY AND STRUCTURE OF GSK3 I. Human Isoforms of GSK3 and Homologs in Other Organisms GSK3 was first cloned in 1990 and is known to exist in eukaryotes in two isoforms, α and β , which are encoded by two independent genes. These genes encode two proteins with molecular weights of 51 and 47 kDa, respectively. The two genes are located on separate chromosomes in humans: the cytological location of GKS3α is 19q13.2, whereas GSK3β is located on 3q13.3 . GSK3α and GSK3β share an overall sequence identity of approximately 85%, but are significantly more homologous within the catalytic domain, where they exhibit a sequence identity of 93%. The predominant difference between the two isoforms is the attachment of a long 83-residue, mostly poly-glycine, tail to the Nterminus of the α-isoform. Despite this unusual structural variation, in prior published studies, purified recombinant GSK3α and GSK3β are stated to exhibit similar biochemical properties as well as similar substrate specificities . In our experience, however, the β-isoform, expressed in bacteria or in insect cells, is far more stable for in vitro studies; the αisoform is much more difficult to over-express, purify, and store, and has a shorter half-life when enzymatic activity is monitored. Studies have shown that the two genes are variably expressed in different mammalian tissues . This differential expression may result from differential levels of transcriptional and translational regulation for the two isoforms. The biological significance of these differences in isoform sequence and structure, as well as the differences in Wagman et al. isoform expression, is unknown. Recently, a splicing variant of the β-isoform has been isolated from the brains of mice, rats, and humans . This variant has a 14 amino acid insert in the C-terminal domain of the protein. The nature and effect of this insert on protein structure and function is not currently known. Like many other kinases, GSK3 activity is both activated and repressed by various phosphorylation events. In other kinases, these phosphorylation events act in an almost Boolean manner, and GSK3 is no exception. Phosphorylation of an N-terminal serine in GSK3 (Ser21 in the α-isoform; Ser9 in the β-isoform), which, in insulin signaling, occurs via by the upstream protein kinase B/Akt, results in inhibition of the enzyme. This inhibitory event can be reversed by protein phosphatase 2A, which has been shown to return GSK3 to an active state . In addition to this inhibition event, GSK3 can also be activated via phosphorylation. Auto-phosphorylation of a tyrosine in the activation segment (Tyr279 in the α-isoform; Tyr216 in the β-isoform) increases enzymatic activity moderately. Dephosphorylation of this tyrosine by tyrosine phosphatase reduces its enzymatic activity [25, 26]. The molecular basis for these inhibition and activation events will be discussed in a following section. There are numerous GSK3 homologs in other organisms and GSK3 has been shown to be widely conserved throughout evolution. Each of these homologs typically segregates towards showing stronger identity to one of the two human isoforms, α or β. GSK3 homologs have been identified in mammals (rats, mice, and others), fish (zebrafish), invertebrates (nematodes, fruit flies, sea urchins, and others), parasites (Plasmodium malariae), plants (Arabidopsis, rice, tobacco, and others), fungi (baker’s yeast and fission yeast), and slime molds . The homology of each of these homologs towards one of the two human isoforms varies significantly from organism to organism, but the sequence identity within the catalytic domain does not fall below 54%, while the overall sequence identity does not fall below 45%. The role of these homologs is also different between species. While the role of GSK3 in mammals and other higher organisms such as fish is strongly conserved and centers around the Wnt signaling and the modulation of glycogen metabolism, as one moves down the evolutionary tree, the role of GSK3 in the cellular milieu changes. This can be seen in sea urchin, where GSK3 is implicated in the establishment of the animal-vegetal (A-V) axis during the early development of the urchin [28, 29]. Further down the evolutionary tree, in the slime mold Dictyostelium discoideum, which possesses a single GSK3 homolog sharing a 70% sequence identity with human GSK3β, the GSK3 homolog plays a critical role in specifying cell fate and controls the differentiation of slime mold cells into either spore or stalk cells [30, 31]. An exhaustive listing of the variation of each homolog’s role in its organism’s biochemistry is beyond the scope of this review, but it is clear that the role of GSK3 homologs shows significant inter-species variation as one moves beyond mammals. II. Enzymology of GSK3 Both isoforms of GSK3 possess a similar enzymatic mechanism: the transfer of the γ-phosphate from ATP to either a serine or threonine, preferentially four residues N- Discovery and Development of GSK3 Inhibitors terminal to a previously phosphorylated serine or threonine (Fig. 1). The preference of GSK3 for substrates with a ‘priming’ phosphorylation event is rare among characterized kinases. Many GSK3 substrates, such as glycogen synthase, often have repeats of -S/T-XXX-S/T- that are phosphorylated in rapid succession [41, 78] once a priming phosphorylation event occurs at the C-terminal serine or threonine within the repeats. This priming event is triggered by another kinase, such as casein kinase 2 (CK2) in the case of glycogen synthase . It should be noted that not all GSK3 substrates require this priming phosphorylation: some proteins involved in the Wnt signaling may not require prior priming to be GSK3 substrates. In such circumstances, the overall structure and local amino acid sequence of the substrate molecule may overcome the need for a priming phosphate (see, for example, the phosphorylation of APC shown in Fig. 1C). The structural basis for the enzymatic mechanism and the need for a priming phosphorylation event will be discussed in the section that follows. GSK3-mediated phosphorylation appears to always lead to inhibition of the substrate: phosphorylation of a substrate by GSK3 has never been shown to be an activating event. III. Overall Architecture of GSK3 and the Structural Basis for Catalysis, Activation, and Inhibition There is currently only structural information for human GSK3β in the form of five crystal structures, four of which Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1107 have been published in the literature, and one of which has been published as a patent application [33-37]. Representative structures are shown in Fig. 2. The overall structure and fold of the human α-isoform and other homologs of this enzyme family should be extremely similar given the strong sequence identity. These crystal structures reveal that GSK3 shares the canonical fold typically seen in serine/threonine kinases and is comprised of an N-terminal β-sheet lobe and a C-terminal α-helical domain . The N-terminal domain forms an incomplete β-barrel and is comprised mainly of seven anti-parallel β-strands as well as one α-helix, the ‘αC’ helix that is responsible for maintaining the nucleotide binding site and activation segment in a catalytically active state. The C-terminal domain is comprised completely of helices and associated loops. The activation segment, which contains Tyr-216, is part of the C-terminal α-helical domain, as is a majority of the substrate-binding site. The ATPbinding site, the catalytic site that encompasses the catalytic machinery, and the substrate-binding site are formed at the interface of the N- and C-terminal domains. This active site ‘channel’ between the two domains measures approximately 22 Å by 13 Å by 15 Å (giving a total approximate volume of 4290 Å3 for the entire active site). By its very nature, the active site of GSK3 is not subdivided into convenient component pockets, but rather is contiguous, with each area melding into the other. An exception is the structural feature responsible for binding the C-terminal priming phosphate group: the oxyanion-binding pocket. This is a structural Fig. (1). For most substrates, GSK3 (either α- or β- isoform) preferentially binds to-and phosphorylates-substrates with a ‘priming’ phosphate four residues to the C-terminus of the serine or threonine residue to be phosphorylated. Many GSK3 substrates have several instances of appropriately placed serine or threonine repeats that are progressively phosphorylated once the far C-terminal residue is phosphorylated (or ‘primed’) by another kinase. Examples of this type of site are shown in (A) and (B). (C) illustrates the GSK3β phosphorylation sites for APC protein. Three more homologous repeats of this type exist on APC protein. Experiments have shown that a priming phosphorylation is not required for subsequent GSK3β phosphorylation . The numbers to the right and left of each particular sequence denote the starting and ending amino acid number of that sequence within the protein in question. 1108 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. Fig. (2). GSK3β structures. (A) Crystal structure of active, phosphorylated Tyr-216, GSK3β. The structure shown is done by Bussiere and colleagues, and is similar to the structures deposited in the Protein Data Bank under accession codes 1H8F and 1I09, which are structures of the less-active, unphosphorylated kinase. The N- and C-terminal ends of the protein are identified, as is the active site, which is denoted by an ‘A’. (B) Crystal structure of active GSK3-β in complex with an axin fragment (PDB accession code 1O9U). (C) Crystal structure of active GSK3β in complex with FRATtide, a fragment of the FRAT protein (PDB accession code 1GNG). Note that axin and FRATtide share the same binding site. feature common to all GSK3β structures and is formed from two arginine residues (Arg-96 and Arg-180) and two lysine residues (Lys-94 and Lys-205) which are arranged in such a way that, in conjunction with local secondary structure, they create a small, highly electropositive, 125 Å 3 pocket ideal for binding a phosphate group. A model of the possible binding mode of primed substrate is shown in (Fig. 3). In support of this structural evidence, mutation of residues within this region greatly reduces the selectivity of GSK3β for substrates with a C-terminal phosphate at the N+4 position . This pocket has been proposed as being suitable for targeting in drug discovery, as it is unique to GSK3 and would enable one to target specific biological responses and disease states [48, 78]. For example, in glucose homeostasis GSK3 is required to recognize ‘primed’ substrates such as glycogen synthase, while in other pathways (such as the Wnt pathway, which will be discussed below), both primed and un-primed substrates are recognized by GSK3 and the primary point of control is the activation of another distinct multi-protein complex . However, recent data has shown that GSK3 mediated phosphorylation of β-catenin is dependent on a priming event by casein kinase-1 α-isoform [40, 41]. Therefore, it is unlikely that compounds that target this site will be directed against a specific disease state. Another drawback is that any molecule designed to bind tightly within this site would most certainly need to be highly negatively-charged, which might lead to undesirable ADME properties for the compound. Proceeding from the oxyanion binding pocket towards the active site (Fig. 3), molecular modeling would predict that the substrate makes a majority of its interactions with the C-terminal domain, although there is the distinct possibility that GSK3 is capable of recognizing structural information at the tertiary level and, therefore, that recognition of substrate is dispersed throughout the molecule and would include recognition by the N-terminal domain as well. Based on structural work with other serine/threonine kinases, as well as the crystal structure of GSK3β, a model of ATP bound in the GSK3β active site can be constructed which predicts that the bound ATP would be stabilized by multiple interactions. First, the adenine ring would form the canonical ‘donoracceptor’ series of interactions, with the extracyclic amino group donating a hydrogen bond to the carbonyl of Asp-133 and the N7 nitrogen of the adenine accepting a hydrogen bond from the amine of Val-135. Additionally, the α- and Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1109 Fig. (3). Structural features of GSK3β. (Α) The active site has several discrete areas: the oxyanion binding pocket, responsible for recognizing the priming phosphate; the phosphotyrosine recognition site, composed of two arginines; and, finally, the active site, responsible for binding substrate and ATP, as well as catalyzing the reaction. (B) The oxyanion binding pocket is made up of two arginines and two lysines, appropriately placed to create a small pocket with a strong electropositive potential. (C) The phosphotyrosine recognition site is composed of two arginines that interact with the negatively-charged phosphotyrosine (Tyr216 in GSK3β), thereby preventing the tyrosine from swinging into the active site and blocking the binding of substrate. Binding of substrate places a serine or threonine adjacent to both the γ-phosphate of ATP (here modeled as AMPPNP) and the catalytic base (Asp-181). Activation of the catalytic base leads to Sn-2 type nucleophilic attack on the γ-phosphate. The transition state is stabilized by Lys-183. γ-phosphates of the ATP would be positioned properly to be stabilized by the Mg2+ ion bound to the ATP itself. The Mg2+ ion is, in turn, predicted to be stabilized by interactions with the side-chains of Asn-186 and Asp-200. With the substrate bound, first at the oxyanion binding pocket and subsequently at various other contact points, the model would predict that the reaction proceeds via activation of the substrate serine adjacent to the ATP through the abstraction of a proton via the catalytic base, in this case, Asp-200. The activated serine or threonine at the N+4 position relative to the priming phosphate would then execute an Sn-2 attack on the γ-phosphate resulting in a phosphorylated serine or threonine and ADP molecule. The transition state of such a reaction is negatively-charged, hence the positioning of the positively charged Lys-85 directly above the site of attack to stabilize the transition state . The proper positioning of Lys-85 is ensured by a stabilizing electrostatic interaction between this lysine and Glu-97, which resides on the αC helix. As mentioned previously, GSK3β can be activated by phosphorylating Tyr-216, thereby increasing its enzymatic activity several hundred-fold. How does this activation work? Current structural information shows that phosphorylation of Tyr-216 and subsequent electrostatic interaction of the negatively-charged phosphotyrosine with the positively-charged Arg-220 and Arg-223 residues swings the phosphotyrosine out of its ‘default’ position to a location where it is no longer blocking the protein substrate’s access to the active site. The default position for the un-phosphorylated tyrosine, on the other hand, places the tyrosine in a position to prevent the binding of protein substrate in the active site. Other than this major structural rearrangement, the remaining structural differences between these two states are relatively minor. This mode of inhibition is contrary to the typical mode of inhibition seen in many closely related kinases where, as in GSK3, phosphorylation of an amenable residue within the activation segment activates the kinase. In other kinases of this type, the activating phosphorylation and subsequent interactions between the phosphorylated residue(s) and the protein results in an ordering of the activation segment leading to optimal positioning of both catalytic residues and residues responsible for binding of substrate in the active site for maximal activity . In GSK3, however, phosphorylation of Tyr-216 reorients this single side-chain relative to the 1110 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 active site, but causes no major changes in the conformation of residues within the activation segment . The mode of phosphorylation-based inactivation of GSK3 by the upstream kinase protein kinase B/Akt at the Nterminal serine (Ser-21 in the α-isoform and Ser-9 in the βisoform) is also hypothesized to occur by a conformational change, but one of a more dramatic nature [36, 48]. In the GSK3β crystal structures, the immediate N-terminus containing the inactivating phosphorylation site is disordered and is not represented in the electron density. Experimental evidence based on competition experiments with peptides derived from the N-terminus, as well as molecular modeling, supports the model that phosphorylation of this N-terminal serine transforms the N-terminus into a pseudosubstrate for the kinase [33, 45]. The model further suggests that in the inhibited form of GSK3, the phosphorylated N-terminus is positioned into the active site with the phosphoserine positioned in the oxyanion binding pocket and a non-reactive amino acid (alanine the case of the α-isoform; proline in the β-isoform) positioned at the N+4 residue position adjacent to the γ-phosphate of ATP. Such positioning of the N-terminus would readily block access to the active site. A crystal structure of the inactivated form of either the α- or β- isoform has yet to be published or disclosed, but a model is presented in Fig. 4. Wagman et al. ANTI-TARGETS OF GSK3 The true challenge of kinase drug discovery and design lies in developing bioavailable, selective, and potent inhibitors [46, 47]. A high degree of selectivity may be required to prevent any given potent compound from having underlying toxicities in vivo. The high degree of three-dimensional structural fold and sequence homology conservation seen between serine/threonine kinases makes attaining this high degree of selectivity a difficult task. To aid in the design of compound specificity in a kinase drug discovery project, one must consider which kinases are ‘anti-targets’ of the kinase of interest. In most cases, an anti-target will almost always be a kinase with significant sequence and structural homology to which lead compounds will also bind. Some project teams initially identify potential anti-targets based on sequence homology, molecular modeling and threading, and by their roles within a cell . Other project teams rely solely on an experimental approach, where initial lead compounds are screened against a broad panel of diverse kinases and cross-reactivity patterns are noted and analyzed for each particular chemical scaffold. It is our opinion that a combined approach is the most appropriate. Any suspected anti-targets identified through bioinformatics or molecular modeling must be confirmed experimentally. Of course, this is not always possible due to the difficulty in overexpressing, Fig. (4). Model of inactivated GSK3β (phosphorylated at Ser-9). Current experimental data supports a model wherein the phosphorylated Nterminus (Ser-9 in GSK3β) acts as a pseudo-substrate for the kinase. The phosphoserine at this position inserts into the oxyanion-binding pocket, thereby positioning a non-reactive residue (a proline in the case of the β-isoform) into the active site, thereby blocking the active site and inhibiting the kinase. This model was constructed using the crystal structure of active GSK3β and structural information from various other kinase crystal structures. Discovery and Development of GSK3 Inhibitors purifying, and storing some kinases, particularly ones which, while identified as part of the kinome, have yet to be expressed, purified, and characterized. Once a panel of significant anti-targets has been confirmed experimentally, additional possible anti-targets can be extrapolated and identified using computational methods. One must take note that an anti-target will be determined predominantly by the particular chemical scaffold(s) under investigation and secondly by the degree of homology between the target of interest and potential anti-targets. Differences in the chemical structure of the compound will combine with the differences in the structural and chemical makeup of the kinase active site to which the compound binds, leading to a different selectivity profile for each chemical scaffold. In addition, one must be aware that, in most cases, the ATP binding pocket will share a higher homology between kinases than the substratebinding pocket(s) or other available pockets, making these other pockets of strategic importance for kinase drug discovery. With this said, however, it should be further noted that currently drug discovery and design directed outside the ATP-binding pocket of a kinase is far more challenging. However, there have been some successes in this area, most notably the development of Gleevec, which was revealed to have a novel binding mode to the inactive form of Abl kinase via an allosteric mechanism . There are reports of other successes using this particular approach, including development of an allosteric P38 MAPK inhibitor [50-52]. Finally, an interesting approach has been taken by several groups who have developed chemical scaffolds known as ‘bisubstrate’ inhibitors. These bisubstrate inhibitors access both the ATP and substrate binding pockets; the drawback to such compounds, despite their potency, is their relatively large size when compared to inhibitors which access only one binding pocket within the active site [54, 55]. The bioavailability and ADMET properties of such scaffolds remain to be determined. What selectivity against anti-targets should be achieved by a kinase therapeutic? This is a difficult question. To some extent it depends both on the anti-target and its cellular role and on the therapeutic goal of the drug discovery project. For example, if one were developing kinase inhibitor as a cancer therapeutic, one might be more tolerant of compound crossreactivity and potential toxicities. However, as the potential indications for a GSK3 inhibitor would most likely be as a therapeutic for the treatment of diabetes or for the treatment of Alzheimer’s disease or a related neuropathology, one would be seeking a more stringent selectivity profile and a compound with no toxicity. For our GSK3 project, we sought a minimum of several hundred-fold selectivity between our GSK3 lead compounds and confirmed anti-targets. By combining synthetic efforts with molecular modeling and broad kinase screening, we were able to develop over a thousandfold selectivity between a compound’s GSK3 activity and its activity in anti-targets. Of course, the guidelines and approaches followed to identify anti-targets and the desirable specificity to be achieved will vary from effort to effort. What are the potential anti-targets for a GSK3 drug discovery project? Again, as discussed above, to a large extent this question is dependent on the chemical scaffold of a lead compound. It is possible, however, to identify broad classes of anti-targets based on structural and sequence Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1111 homologies. The crystal structures of GSK3β showed that there is a structural relationship between GSK3, the various cyclin-dependent kinases (CDKs), and the mitogen-activated protein kinases (MAPKs). Because of this homology, any kinases in these two classes are possible anti-targets for any chemical scaffold. In particular, sequence homology and threading of the active site region suggest that the active sites of cyclin-dependent protein kinases 2 (CDK2/p33) and 3 (CDK3) are most homologous to the GSK3β active site [56, 57]. However, these broad classes do not represent the only possible anti-targets. For example, our GSK3 project team empirically identified CHK1 as an anti-target for one of our second-generation chemical scaffolds, yet it was not a significant anti-target for our first-generation compound (Data to be published elsewhere). This identification would not have been made on the basis of structure and sequence homologies; instead it had to be identified experimentally. This cross-reactivity arose to due an electronegative patch present in both the GSK3 active site and the CHK1 active site that interacts with a positively charged moiety within the chemical scaffold. The electronegative patch in the active site arises not only from the primary amino acid sequence, but also from main-chain carbonyl orientations that cannot be predicted on the basis of amino-acid sequence comparisons, thereby illustrating the often-arcane mechanism of crossreactivity. By using structure-based drug design, the project team was able to design in over one-thousand fold selectivity against CHK1 into this chemical scaffold, thereby illustrating the importance of molecular modeling and structural biology in kinase drug discovery. GSK3, MULTI-PROTEIN COMPLEXES, AND THE WNT SIGNALING PATHWAY Aside from GSK3 roles in the insulin signaling pathway, tau protein hyperphosphorylation, and other physiological events where it acts alone by catalyzing the phosphorylation of select serine and threonine residues, a portion of the cellular GSK3 forms a component of a multi-protein complex involved in the Wnt signaling pathway. GSK3 is able to perform its role in both of these pathways with no apparent cross talk between the pathways . This multi-protein complex is comprised of GSK3β, axin, β-catenin, and APC (adenomatous polyposis coli) protein . GSK3α has not been identified as being involved in this multi-protein complex. Wnt signaling pathways are mediated by secreted glycoproteins known as Wnts. These Wnts are paracrine signaling molecules and activate numerous signaling cascades inside target cells [59, 60]. In the absence of an active Wnt signal, the GSK3β in the complex is active and phosphorylates each member of the complex at various positions in the constituent proteins [61, 62]. These phosphorylations potentiate the action of two of the proteins within the complex: the phosphorylation stabilizes axin and strengthens the interaction between β-catenin and APC protein . At the same time, however, the phosphorylation targets β-catenin for ubiquitin-mediated proteolytic degradation by the proteasome (Fig. 5) [64, 65]. In the presence of an active Wnt signal, the activity of GSK3β in the multi-protein complex is inhibited by the disruption of the GSK3β:axin interaction by another multi-protein complex, which contains at least two proteins. These proteins, known as DVL (disheveled) protein 1112 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. Fig. (5). GSK3β’s role in the Wnt pathway. In the absence of Wnt signaling via the binding of Wnt to its receptor, a multi-protein complex scaffolded by APC and Axin promotes phosphorylation of β-catenin by GSK3β. APC and Axin are also phosphorylated by GSK3β in this complex; some models also propose that casein kinase 1 (α− or ε−isoform) and other proteins are also members of this complex. In this schematic, they have been omitted to simplify the model. The phosphorylated β-catenin is degraded by ubiquitin-mediated proteolysis by the proteasome. It should be noted that this pathway, which leads to degradation of b-catenin is the basal state. Wnt binding to the 7-TM ‘frizzled’ receptor activates another multi-protein complex comprised of DVL protein, FRAT protein, and perhaps other as-of-yet unidentified protein members, which then bind to GSK3β and inactivate it, displacing the other three proteins, APC, Axin, and β-catenin. This prevents phosphorylation of β-catenin by GSK3β and, thus, prevents subsequent degradation of the β-catenin by the proteasome. βcatenin accumulates both in the cytosol and the nucleus, where it serves as a trans-activator in complex with Lef/TCF DNA-binding proteins leading to activation of various developmentally critical target genes. and FRAT (an acronym for ‘frequently rearranged in advanced T-cell lymphomas’) protein, bind directly to GSK3β and inhibit its function as well as its binding interaction with axin . The molecular mechanism that regulates binding of this complex directly to GSK3β is unknown. The resulting inhibition of GSK3β stabilizes the β-catenin in the cell, which leads to its steady accumulation and eventual translocation to the nucleus, where it interacts with transcription factors within the T-cell factor (TCF) family . This interaction triggers the transcription of genes that are required for determination of cell fate during embryogenesis. This event is also likely to be involved in regulating the expression of other genes involved in regulating events in adult tissues. The formation of the GSK3β:axin:β-catenin:APC complex would explain the dichotomy between the roles of GSK3β in the insulin and Wnt signaling pathways. Sequestration of GSK3 into the complex facilitates access to the primary substrate in the Wnt pathway: β-catenin. The formation of the complex might also prevent protein kinase B/Akt from accessing the N-terminal inactivating serine, thereby preventing GSK3β from being inactivated while it is in the multi-protein complex. Direct structural determination of the complete GSK3β: axin:β-catenin:APC protein complex has yet to be achieved, but components of this multi-protein complex have been determined by X-ray crystallography. These include the multiple structures of GSK3β (discussed previously), structures of β-catenin in complex with other members of the Wnt pathway, and finally, a structure of a fragment of the APC protein in complex with β-catenin [68-70]. There are currently only two protein complex structures involving GSK3β and another member of the Wnt-pathway, the first being a structure of active GSK3β (Tyr216 is phosphorylated) with a peptide fragment of the FRAT protein known as the ‘FRATtide’, which has been shown experimentally to inhibit GSK3 in manner equivalent to the intact FRAT protein . Discovery and Development of GSK3 Inhibitors This structure shows that the fragment of FRAT, of which residues 188-226 are visible in the electron density maps (comprising approximately 17% of the total protein), forms a helix-turn-helix that interacts with GSK3β at the C-terminal α-helical domain of the protein. The interaction between GSK3β and the FRATtide is primarily mediated by hydrophobic side-chain contacts along the face of the Cterminal FRATTide α-helix that are inserted into a hydrophobic patch between a loop (residues 285-299) and an α-helix (residues 262-273) on the C-terminal domain of GSK3β. Structural and biochemical details of FRAT activation, which may involve an activating event such as phosphorylation, are not currently known. The second crystal structure is a structure of GSK3β in complex with a 19 amino acid minimal binding segment of human axin [37, 72]. This structure reveals that the interaction between GSK3β and the minimal axin binding segment, which forms an α-helix in the crystal structure, is very similar to the interaction between GSK3β and the FRATTide: the interaction is mainly through hydrophobic contacts at the same hydrophobic patch as is seen in the previous structure. Based on this data, the binding of FRAT or axin to GSK3β would be mutually exclusive, as both share the same binding region. This is in agreement with the current model. These two structures are presented in Fig. 2. GSK3 SMALL MOLECULE INHIBITORS AND DRUG DISCOVERY PROGRAMS While our understanding of the prominent regulatory role of kinases in cell signal transduction has grown remarkably over the last 20 years, our understanding of the intricate and often interdependent cellular kinase control mechanisms has recently undergone a renaissance with the increasing availability of potent and selective small molecule kinase inhibitors. Pharmaceutical research that has recently yielded kinase inhibitors of clinical interest in the treatment of cancer and chronic inflammatory diseases, among others, has also created a bounty of kinase-specific molecular probes which have been successfully exploited in the study of cell signaling . Encouraged by a growing number of kinase drug targets and an increasingly sophisticated knowledge of kinase activity and modulation, drug discovery research has taken up the challenge of finding safe and efficacious kinase inhibitors for the treatment of chronic conditions such as diabetes . A promising drug target, GSK3 has been the focus of intensive medicinal chemistry efforts not only for insulin resistance, but also for Alzheimer's disease, stroke and bipolar disorders [2, 9, 75]. In this section, research which has yielded potent and selective inhibitors of GSK3 will be highlighted along with a summary of the classes of small molecule inhibitors which appear in the patent literature. For recent reviews see [74, 76, 77]. Emphasis will be placed on structurally novel compounds and small molecule research programs that have generated in vitro SAR, cellular potency and biological studies. Before the availability of selective inhibitors, initial studies of GSK3 were conducted using lithium. The effects of lithium on developing organisms were traced to GSK3 inhibition and shown to mimic the effects of Wnt signaling [78-80]. Since lithium is used in the treatment of bipolar disease and depression, a connection was made between Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1113 GSK3 and possible molecular mechanisms leading to these mood disorders [81-83]. The potential association between GSK3 and mood-stabilization was further enhanced by the discovery that valproic acid, used in the treatment of bipolar disorder, also activated the Wnt signaling pathway . The research surrounding lithium and valproate as modulators of the Wnt pathway, their mechanistic and biological characteristics and utility in bipolar disorder has been accounted in detail . Recently, however, histone deacetylase has been identified as the target for valproate and the basis of this compound's potent anticonvulsant and mood-stabilizing action . Further research will be needed to evaluate the involvement of GSK3 in valproate mediated physiological responses . LITHIUM Lithium has been shown to reduce the phosphorylation of tau protein in cells at therapeutic concentrations in a manner consistent with inhibition of GSK3 . Lithium has also been shown to protect neuronal cells from apoptosis caused by the accumulation of fibrillary β-amyloid which can be explained via lithium's action as an inhibitor of GSK3 and provides evidence that GSK3 might be a viable target for the prevention of neurodegenerative disorders such as Alzheimer's disease. The implication of GSK3's involvement in the pathology of Alzheimer's disease through interactions with protein tau, presenilin 1, the amyloid-β peptide, the amyloid precursor protein, and acetylcholine and proapoptotic mechanisms has prompted a great deal of interest in these pathways and the possible use of GSK3 inhibitors for the treatment of many neurodegenerative diseases including stroke and acute brain trauma [89-92]. As an inhibitor, lithium exerts a direct effect on GSK3 having an IC 50 of 2mM under conditions thought to approximate the intracelluar environment i.e. a free concentration of Mg2+ (0.5mM) and isotonic KCl 150mM . Lithium does affect GSK3 activity to a greater extent then other kinases tested under the same conditions, however lithium is not highly potent or selective. At several multiples (3-7 times) of the IC50 of GSK3, lithium inhibited casein kinase 2 (CK2), p38regulated/activated kinase (PRAK) and MAPK-activated protein kinase 2 (MAPKAP-K2). It is possible that some of the effects attributed to GSK3 from studies using lithium may in part be from alternate kinase targets. For example, before glycogen synthase (GS) can be inhibited through the kinase action of GSK3, it must first be primed by phosphorylation by CK2. The ambiguity associated with lithium's cellular activity underscores the need for selective, potent and cell-permeable probes of kinases (and GSK3) to resolve their independent contributions to cellular regulation. GENERAL KINASE INHIBITOR POTENCY AND SELECTIVITY SURVEY As part of a greater effort to characterize the mechanism of action and specificity of 42 commercially available kinase inhibitors against a large panel of some 30 different protein kinases, several small molecules were found to significantly inhibit GSK3 [93, 94]. While this study was instrumental in identifying GSK3 inhibitors, it also defined the selectivity patterns of a variety of ATP competitive kinase inhibitor compounds and classes of molecule, including some natural 1114 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 products. In general, this selectivity and potency information helps guide the application and interpretation of studies undertaken with these inhibitors. Many compounds inhibited more kinases than their expected target and even the most selective molecules inhibited at least one additional kinase along with their target. It is, therefore, critical to take these findings into consideration when designing new experiments or interpreting existing whole cell or in vivo studies with these classes of kinase inhibitor. In the first phase of the study, Ro 318220 (1), which was designed and used as a PKC inhibitor, was found to be the most potent inhibitor of GSK3 (IC50 = 38nM) among a group of bisindolylmaleimides. All assays were run at ATP concentrations of 0.1mM, and inhibition is expressed as the percent activity as compared to 100% activity using the appropriate substrate. Other bisindolylmaleimides including Bis-1, Bis-3, Bis-4, Bis-5, Bis-8, Bis-10, Go6976, K252c, KT 5720 and UCN1 were assayed. Of these only Bis-1, Bis-3, Bis-4, Bis-8, Bis10 and Go6976 had activities between 21 and 50% of normal full activity at 1uM. None were highly selective for GSK3. Three additional compounds (Rottlerin - 13% activity at 20uM, KN62 - 38% activity at 10uM and Quercetin - 30% activity at 20uM) were found to weakly inhibit GSK3 among other kinases . SP600125 and KT5823 were also found to have weak activity . O H N N Wagman et al. inactive . The higher IC50 reported in these studies may be explained by the lower ATP concentrations used in the assays, as IC50 increases with ATP concentration for ATPcompetetive inhibitors such as these. While in most cases potent CDK inhibitors are active against GSK3, selectivity was found with two examples from the indirubin class; 5, 5'dibromoindirubin (3) was selective for GSK and 5-SO3Naindirubin-3'-monoxime (4) was more active against the CDKs. The combination of known selectivity in this scaffold and the X-ray crystal structure of indirubin-3'-monoxime (2) bound in the active site of CDK2 may indicate productive areas where these molecules might be further modified to enhance their selectivity toward GSK3. Since the parent compound indirubin (5) has poor bioavailability and has shown some gastrointestinal effects, the scaffold would also have to be chemically modified to improve PK and tolerability for use in a chronic application . R3 R2 R1 N H NH O 2: R1 = R3 = H; R2 = NOH 3: R1 = R3 = Br; R2 = O 4: R1 = H; R2 = NOH; R 3 = SO 3Na 5: Indirubin: R1 = R3 = H; R2 = O O N Me NH S NH 2 1: Ro 31-8220 INDIRUBINS In the second part of the study, traditional CDK inhibitors reported to have GSK3 activity were confirmed. Indirubin-3'-monoxime (2), which is a component of the traditional Chinese medicine Danggui Longhui Wan, has been used in the treatment of chronic diseases such as leukemia . It is known as a potent (IC50 = 50-100nm) ATP competitive inhibitor of CDK1, CDK2 and CDK5 and has been found to initiate cell-cycle arrest as expected from a CDK inhibitor . In the survey study, indirubin-3'-monoxime (2) was active against GSK3 (IC 50 = 190nM), CDK2 (IC50 = 590nM) and several other kinases, such as AMP-activated protein kinase (AMPK) and serum- and glucocorticoid-induced kinase (SGK) . It is not surprising that an ATP competitive inhibitor of the CDK family would also be an inhibitor of GSK3 since these kinases are closely related and have similar active sites (see above). In a separate study, a variety of indirubins, including indirubin-3'-monoxime (2), were identified as potent inhibitors of GSK3 (IC50 = 5-50nM) and CDKs, while other structurally related indigoids were PAULLONES Members of the naturally occurring family of benzazepinones called paullones (6: paullone) were found to be potent inhibitors of GSK3 with fairly good selectivity against the panel of kinases assayed. Kenpaullone (7) (IC50 = 230nM) and alsterpaullone (8) (IC50 = 110nM) were both potent ATP competitive inhibitors of GSK3, but also inhibited LCK and CDK2/cyclin A. Alsterpaullone (8) was actually more potent against CDK2/cyclin A (IC50 = 80nM) than GSK3. Although, the IC50's for both compounds were within 3 fold for all three of these kinases . Similar to previous well known kinase inhibitors, paullones were found to have anti-cancer activity which was linked to their potency against CDK1/cyclin B [98, 99], and later to their ability to inhibit GSK3, CDK2 and CDK5 . While not inhibiting any isoform of PKC, MAPKK or c-raf, alsterpaullone (8) did inhibit CDK1/cyclin B, CDK2 cyclinA, CDK2 /cyclin E, CDK5/p35 and GSK3α and β. The anti-tumor effects of alsterpaullone (8) are most likely due to inhibition of CDK1/cyclin B. The suppression of tau phosphorylation by either alsterpaullone (8) or kenpaullone (7) may be due to GSK3 and/or CDK5 inhibition. Alsterpaullone (8) also suppresses the phosphorylation of DARPP-32 by CDK5/p25 which may reduce apoptosis signals in neurons and would be helpful in the treatment of Alzheimer's disease. SAR from a diverse set of 56 paullones showed that substitution of small groups at the C-9 position increased potency. It was hypothesized that incorporating a hydrogen bond acceptor at C-9 would increase the binding Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1115 pocket affinity by interaction with a proximal molecule of water identified in the crystal structure of CDK2. The addition of a nitro group at C-9 did improve the IC50 of alsterpaullone (8) (IC 50 CDK1 = 35nM) over the parent paullone (IC50 CDK1 = 3uM) , however the selectivity between GSK3, CDK1 and CDK5 was lost . A small lipophilic group at C-9, such as a chloro or bromo group, increased the potency and selectivity for GSK3. The pyrrole-like NH at position 12 could be alkylated with many small groups while still maintaining potency. Interestingly, a methyloxycarbonylmethyl group on position 12 nearly obliterates CDK5 binding and improves GSK3 selectivity (IC 50 GSK3 = 75nM, CDK1 = 1.4uM, CDK5 = 350uM). Methoxy groups at C-2 and 3 are also tolerated, but do not improve the kinase selectivity. 5 H N O 3 2 12 HN 9 R 11 6: R = H Paullone 7: R = Br Kenpaullone 8: R = NO2 Alsterpaullone HYMENIALDISINE Hymenialdisine (9) and related metabolites produced by several species of marine sponge represent a novel family of ATP competitive kinase inhibitors [101, 102]. While screening natural products for possible anti-cancer activity, hymenialdisine (9) was found to be a potent inhibitor of CDK1/ cyclin B. Other than cancer applications, interest in these compounds has also been raised due to their anti-inflammatory activity in U937 cells through inhibition of nuclear factor-kB . Hymenialdisine (9) shows potent activity against CDK1/cyclin B (IC 50 = 22nM), CDK2/cyclin A (IC50 = 70nM), CDK2/cyclin E (IC50 = 40nM), CDK3/cyclin E (IC50 = 100nM), CDK5/p25 (IC50 = 28nM), CK1 (IC50 = 35nM) and GSK3 (IC50 = 10nM) with good selectivity versus other kinases such as Erk2, c-raf, MAPKK and the PKCs , and has been filed in a patent application . The chemically related diacetylhymenialdisine (10) and diacetyldebromohymenialdisine (11) are fairly active inhibitors of GSK3 (IC50 = 130 and 160nM), while the close analogues hydantoin (axinohydantoin, 12) and 2-aminoimidazole (Stevensine/odiline, 13) had poor or no activity respectively. Clues to the selectivity of hymenialdisine (9) can be gleaned from the crystal structure of the inhibitor bound to the ATP binding site of CDK2. The guanidine ring of hymenialdisine (9) which seems to be a key component of potency and selectivity is in H-bond proximity to three crystal bound waters in the ATP pocket near Gln131 and Asp145. The hydrophobic bromopyrroloazepine bicyclic ring system of hymenialdisine (9) fits snugly in a hydrophobic pocket making key backbone H-bonds to Leu83 and Glu81. Structure based approaches for increasing the selectivity of these compounds might be possible by studying the differences between the CDK2 and GSK3 crystal structures which are now available. Experimental studies using hymenialdisine (9) show promise for treatment of neurodegenerative diseases. Phosphorylation by GSK3 and CDK5/p35 was suppressed in a dose dependent manner. At high doses, hymenialdisine (9) blocked the phosphorylation of MAP-1B and had an affect on axonal remodeling. Consistent with GSK3's activity against other substrates, hymenialdisine (9) inhibited the phosphorylation of expressed human tau with an IC 50 of ~33nM giving an indication that potent inhibitors of GSK3 and/or CDK5/p35 may find utility in the treatment of Alzheimer's disease . SMALL MOLECULE PHARMACEUTICAL RESEARCH PROGRAMS Highly Substituted Purines, Aminopyrimidines and Aminopyridines Early collaborative research at Chiron in 1996 identified substituted purines (14) as novel synthetic, ATP competitive inhibitors of GSK3 (63% inhibition at 1uM), validating the tractability of the kinase as a target for medicinal chemistry [106, 107], and helping to improve and refine the optimal methods for screening potent small molecules [108, 109]. Further medicinal chemistry research yielded a very potent series of GSK3 inhibitors based on highly substituted aminopyrimidine 15 (W = N) or aminopyridine 15 (W = CH) core O H 2N O NH N N HN O H2N NH HN HN O Br R1 O HN NH NH O O 9: Hymenialdisine Br HN N NH Br O Br HN NH N 10: R1 = Br 11: R1 = H O NH O 12 13 1116 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. rings [110, 111]. Later it was revealed that the core ring could also be an aminopyrazine 16  or a fused bicyclic system 17 wherein a ring was formed between the pictured C-5 and C-6 positions of the core ring . A common feature of this family of analogues is the arenes linked by a bridging ethylenediamine. On one side of the four atom bridge are the substituted pyrimidines and pyridines having a decorated phenyl group in the C-4 position (see structure 15). Substitution at the C-5 position was highly varied, but many groups were capable of making productive first and second shell water contacts on the surface of the protein, as well as possible H-bonding interactions with amino acids at the entrance of the ATP binding pocket. This position also provided a convenient chemical handle to optimize the physicochemical and PK properties of the compounds. The C-4 phenyl ring was typically substituted with small groups, such as halo, methyl and methoxy moieties at the C-2' and 4' positions, although much larger groups were tolerated the 4' carbon which was directed back toward the substrate binding area of the active site. The C-2' substituents were optimally placed in an accommodating, small hydrophobic pocket which greatly enhanced the kinase selectivity of the series . Linked through the C-2 position of the pyrimidine core, the distal nitrogen of the ethylenediamine was typically coupled via SnAr reaction to an activated nitrogencontaining heteroaromatic ring such as, a 5-nitropyridine, 5cyanopyridine or 4-nitrothiazole. Some of the details of the evolution of the aminopyrimidine and aminopyridine series of compounds in the context of a program directed toward novel anti-hyperglycemics have been disclosed [74, 115-117]. This series found it's genesis in a kinase-directed library of 80 pools of 3, 4dihydropyrimidines 18. Each pool contained a mixture of 18 compounds. After extensive GSK3 screening, only one pool furnished modest inhibition activity in the 3-5 uM range. This mixture of 18 molecules continued to be used in biochemical studies where it was noted that over time, the activity of the compounds seemed to be improving. Aware that the 3, 4-dihydropyrimidines could undergo oxidation when exposed to chemical oxidants or air and heat, it was assumed that the fully aromatic 2-aminopyrimidines were responsible for the improved activity of the pool. Upon investigation of the pool using HPLC and MS, it was found that not only had the core ring aromatized, but the residual Rink-resin linker amide on the C-4 phenyl group had dehydrated to a cyano group as represented by the transition from CT 98018 (19) to CT 98016 (20). Thus, when the pool constituents were deconvoluted and individually assayed, CT 98016 (20) was found to inhibit GSK3 with an IC50 of 50nM and had activity in an insulin-responsive CHO cell line. With this serendipitous discovery, the most potent leads in this medicinal chemistry program transitioned from low uM IC50s to nM potencies and provided a highly attractive scaffold for optimization. CF3 HN O O N N N N N H2N X 14 O Y X Y X Heterocycle Heterocycle 4 R1 4 R1 NH N 3 5 5 W 2 N H 1 HN 5 2 N H 1 O NH2 NH2 NH N 3 N 2 N H 1 16 15: W = CH or N O Heterocycle 4 NH N 3 N R2 Y 17 CN NO2 NO2 N O R2 O R1 NH NH N 18 N H N O O NH NH N N H 19: CT 98018 IC50 = 3-5 µM EC50 = >10 µM O O NH N N N H 20: CT 98016 IC50 = 50 nM EC50 = 1.6 µM Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1117 Traditional medicinal chemistry surveys of allowable substitution demonstrated that a linear four atom linker was needed, and an ethylenediamine linker was optimal between the aryl groups. Substitution on the NH proximal to the pyrimidine was tolerated, but an H-bond donor was needed on the distal side to make backbone amide bonds in the ATP pocket. A four atom linker also provided the best alignment between the C-4 phenyl, core pyrimidine and 5-nitropyridine in the binding pocket. Larger or smaller linkers were stericly disfavored, presumably causing collisions between the linker and main chain residues and misaligned the C-4 phenyl in a channel leading to the substrate binding area. Groups such as the C-6 ethyl were removed to lower molecular weight and decrease lipophilicity. The C-5 ethyl ester of CT 98016 (20) was utilized to improve solubility. Unfortunately, while the free carboxylate, basic ester or basic amides at C-5 were potent in the cell free assay, they did not show GSK3 activity in the IR-CHO cell assay which is most likely due to poor membrane permeability (21) . Substituents on the C-4 phenyl group were best tolerated in the ortho and para CN NO2 positions. Smaller groups, such as F, Cl, Br, CF3, OMe, and OCF3, were preferred in the ortho position, while larger groups, such as ethyl, propyl and imidazole, were permitted at the para position. However, the series was optimized using a 2, 4-dichlorophenyl at C-4 which seemed to give improved IC50's. Switching from linear moieties to heterocycles, an Nlinked imidazole, such as CT 98014 (22), gave a notable improvement in the whole cell activity. A second refinement which enhanced the IR-CHO cell EC50 was the discovery a new 6-amino-5-nitropyridine group at the distal end of the ethylenediamine linker. A survey of several hundred heterocycles at the distal linker position yielded only a few compounds with sufficiently potent IC50's (e.g. <100 nM) (Scheme 1). 2-Aminopyridines with electron withdrawing groups at the para position gave the best results. It became apparent that an electron withdrawing group was needed in the para position of the pyridine for optimal binding, for example CT 98018 and CT 98224 in Scheme 1. Crystal structures and modeling show a close approach of this NH to the backbone in the ATP binding pocket . This suggests Cl NO2 Cl NO2 NH2 N O N Cl X N N NH N N N H 21: X = -OH, -OCH2CH2NMe2 -NHCH2CH2NMe2 N 22: CT 98014 N H 23: CT 98024 IC50 = 560 pM EC50 = 387 nM IC50 = 580 pM EC50 = 83 nM IC50 = 30-160 nM EC50 = 3-10 mM NH N N H N H N Cl N NH N N NH2 Cl Cl N Heterocycle N NH N N NO2 CN NO2 N CT 98018 IC50 = 6 nM N H CT 98014 IC50 = 1 nM CT 98224 IC50 = 10 nM N N N N Cl CT 98022 IC50 =201 nM Scheme 1. CT 98032 IC50 = 24 nM NH2 N N N CT 98124 IC50 = 223 nM CF3 CN N Cl N N N CT 98028 IC50 = 87 nM NO2 S N NH2 CF3 N CT 98030 IC50 = 236 nM N CT 98026 IC50 = 381 nM S CT 98132 IC50 = 596 nM 1118 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. that modulation of the pKa of the NH of the amino pyridine group adjusts the strength of an important backbone Hbonding interaction. Application of these modifications lead to highly potent compounds, and prompted the initiation of animal studies. While not very soluble, CT 98014 (22) was used to examine the potential of the series to improve the insulin sensitivity and glucose disposal in a mouse model of diabetes. Following oral dosing at 30 mg/kg (20% Captisol) in db/db diabetic mice, the compound elicited a 25% reduction in blood glucose AUC in an oral glucose tolerance test (oGTT) with a rapid (~30min) onset of action. To improve the solubility and formulatability of the series, the C-5 imidazole was rotated in CT 98024 (23) to provide a H-bond donor, an accessible pKa and sub-nM potency (IC50 = 560pM). CT 98024 (23) was easily formulated in citrate buffered water and was orally efficacious in an oGTT model in db/db mice (30% reduction in glucose AUC at 30mg/kg dose) and ZDF rats (29% reduction glucose AUC at 8mg/kg dose). Unfortunately, CT 98024 (23) suffered from primary and secondary metabolism resulting in a poor half life (t1/2) in rodents (46 min. oral t1/2 in rat). The metabolism issues were address by methylating the easily oxidized imidazole and by replacing the nitropyridine with a 5-cyanopyridine generating an early clinical candidate, CT 99021 (24). The oral half life doubled in rats to 92min. The oGTT showed a 32% reduction in glucose AUC at 8mg/kg dose and an ED 50 of 6mg/kg in ZDF rats with twice-daily dosing. Further iterative cycles of medicinal chemistry and improvements of in vivo characteristics lead to CT 20026 (25), one of Chiron's most advanced predevelopment compounds. Replacement of the metabolically unstable imidazole with a monoketopiperazine improved the solubility and stability of CT 20026 (25) over previous leads. Use of a 2-aminopyridine core in place of a pyrimidine gave a different metabolic profile, as well as improving cell permeability as evidenced in the IR-CHO cell activity (EC50 = 66nM). The PK of CT 20026 (25) was also improved showing longer half-lives in rats (205min), beagle dogs (163min) and cynomolgus monkeys (173min). The oGTT in ZDF rats provided an ED 50 of 10 mg/kg after single administration with an oral bioavailability of ~30%. Despite the documented mutagenic potential of nitroarenes, no geneotoxicity was observed in a full Ames study. Extensive evaluation of this family of GSK3 inhibitors in vitro has established that they are exquisitely selective for Cl the GSK3 ATP binding site even when assayed against structurally similar kinases with high sequence identity like CDK1 and ERK2, the closest homologs with 30% amino acid identity within their catalytic domains . The results of the selectivity panel for CT 98014 (22) and CT 99021 (24) assayed against 20 kinases show 500 to >10, 000 fold selectivity, which is representative of the whole class of inhibitors (Table 1). There was virtually no difference in IC 50 between the isoforms of GSK3α and β, and little difference in inhibition between human, mouse and rat derived proteins. CT 99021 was also evaluated against a panel of 22 standard screening receptors and 23 non-kinase enzymes. The only off-target activity (Ki = 8.3 umol/l) was against phosphodiesterase III. Having established the high degree of selectivity of these compounds, they were used with confidence in studies designed to probe GSK3's role in cellular glucose metabolism. In insulin receptor-expressing CHO-IR cells, GS activity was increased several fold above basal in a dose dependent manner in response to either CT 98014 (22) or CT 99021 (24) (EC 50's of 106 and 763 nmol/l respectively). The higher EC 50 for CT 99021 (24) is consistent with it's higher Ki. Some variation in EC50 between compounds may also be due to differences in cell membrane permeability or protein binding. To demonstrate that these GSK3 inhibitors could function in a more natural cellular environment, CT 98014 (22) was added to primary rat hepatocytes which responded as the CHO-IR cells did with a two- to threefold stimulation of the GS activity. The EC50 of CT 98014 (23) in the rat hepatocytes was 107 nmol/l which is very similar to the EC 50 in CHO cells . It is interesting to note that the GSK3 inhibitors were able to increase the activity of GS to the same or greater extent as insulin might accomplish through suppression of GSK3 activity. The effect of these compounds on insulin sensitivity and improved glucose uptake will be discussed below. MALEIMIDES Staurosporine (25) and structurally related maleimidecontaining molecules, such as Ro 318220 (1) and GF 109203x (25), have consistently demonstrated their utility as kinase inhibitors, especially inhibitors of PKC. As early as 1999, Ro 318220 (1) and GF 109203x (25) were identified as inhibitors of GSK3 β with IC 50's in the low nanomolar and hundreds of nanomolar range respectively . Unfortunately, staurosporine and Ro 318220 (1) (as noted above) are CN Cl NO 2 NH 2 H3 C N Cl N N NH N N N H N N H N Cl NH N O N N H 24: CT 99021 25: CT 20026 IC50 = 5 nM EC50 = 1 uM IC50 = 4 nM EC50 = 66 nM Discovery and Development of GSK3 Inhibitors Table 1. Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1119 Selectivity of Kinase Inhibition by CT 98014 and CT 99021 Kinase species group identity CT 98014 IC50 (nM) CT 99021 IC50 (nM) GSK3-β human CMGC3 100% 0.58 Ki = 0.87nM 6.7 Ki = 9.8nM GSK2-α human CMGC3 91% 0.65 10 cdc2 human CMGC1 30% 3,700 8,800 erk2 human CMGC2 31% >10,000 >10,000 PKC-α human AGC2 22% >10,000 >10,000 PKC-ς human AGC2 19% akt1/PKB human AGC3 21% >5,000 >10,000 p70S6K rat AGC6 22% >1,000 >10,000 p90RSK2 rabbit C6,CAMK6%, 22% >10,000 >10,000 PTK01 19% >1,000 c-src >10,000 tie2 human PTK13 18% >5,000 >5,000 fit1 human PTK14 17% >5,000 >5,000 KDR human PTK14 18% >2,000 >5,000 bFGFRTK human PTK15 20% >1,000 >5,000 IGF1RTK human PTK16 16% >2,000 >10,000 InsulinRTK human PTK16 16% >2,000 >10,000 AMPkinase rat OPK 22% >10,000 pdk1 human OPK 23% >10,000 chk1 human OPK 21% >10,000 >10,000 CK1-ε human OPK 17% >5,000 >5,000 DNAprotein human P13K Low >10,000 PI3kinase human P13K Low >2,000 >10,000 Percentage identity refers to amino acid identity versus human GSK3β within the protein kinase catalytic domain. Abbreviations: RTK, Receptor Tyrosine kinase; CK1ε, Casein Kinase 1 epsilon. The group classification represents the class of kinase from which the particular enzyme derives . not highly specific kinase inhibitors , although staurosporine analogues have been filed as GSK3 inhibitors . Indeed, Ro 318220 (1) may have biological activity against targets other than kinases, such as inhibition of sodium channels . While interesting as biochemical probes, these compounds do not have sufficient selectivity to be used as anti-hyperglycemics. However maleimides are potent kinase inhibitors which might be a good starting point for optimization. Having identified this scaffold by high throughput screening of rabbit GSK3α, SmithKline Beecham generated a matrix of compounds to explore the SAR of GSK3α binding . Maleimide analogues were quickly established with low nanomolar activity against human GSK3α (IC50s 20-50nM) [123-125]. The 3 most potent compounds reported had acidic phenols (27) or carboxylates (28, 29) on the aniline moiety and a nitro group on the aryl ring. Methylation on either the aniline nitrogen (30) or the maleimide nitrogen (31) resulted in a significant loss of potency. Interestingly, the binding pocket did tolerate the use of an indoline (32) in place of an aniline with minimal loss of activity. In the indoline series, the aniline H-bond is not critical, suggesting an alternate binding mode. Five top compounds (including SB-415286 (33)) demonstrated good selectivity when screened against a panel of 20 kinases. As expected from the similarity of the ATP binding pocket, these molecules were equipotent against GSK3α and β. SB415286 (33) was modestly active against RSK-2 and AMPK. The biological activity of the arylindolemaleimide SB216763 (34) and anilinomaleimide SB-415286 (33) (IC50's of 34nM and 78nM respectively) were reported in detail  (arylindolemaleimides were also filed in applications independently ). Both were shown to be highly potent ATP competitive inhibitors of human GSK3α. They were also able to stimulate glycogen synthesis in Chang human liver cells and HEK293 human kidney cells at levels higher than the maximal rate of insulin alone. To investigate the 1120 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. NH H N O O H N O O R2 N Me HN O N N H MeO N R1 H MeHN 26: GF 109203x R1 H N O N 27: R1 = 3-NO2; R 2 = 3,5diCl-4-OH 28: R1 = 2-NO2; R 2 = 4-Cl-3-CO2 H 29: R1 = 3-NO2; R 2 = 4-Cl-3-CO2 H N 25: Staurosporine O O O O O H N O Cl O2N N N O2N HN OH R2 32 30: R 1 = H; R2 = Me 31: R 1 = Me; R2 = H ability of these GSK3-specific inhibitors to induce β-catenin stabilization and accumulation in the cytosol, compounds were assayed in HEK293 cells with a β-catenin-LEF/TCF regulated reporter gene. Both compounds gave a dosedependent response indicating a direct inhibition of GSK3 and increased β-catenin accumulation in this in vitro system. Additional studies show that these compounds can mimic the action of insulin by suppressing gene transcription of key enzymes (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) in the gluconeogenic pathway . SB216763 (34) and SB-415286 (33) are also able to protect both central and peripheral nervous system neurons from death induced by abrogation of PI3 Kinase activity using the PI3 Kinase inhibitor LY-294002 (35) in culture . The neuronal protection correlated with the inhibition of GSK3 and the modulation of tau phosphorylation and β-catenin protein levels . These results, combined with recent studies demonstrating that FRAT1 overexpression is neuroprotective, emphasize the potential value of selective and potent small molecule GSK3 inhibitors, not just for chronic diseases such as diabetes and Alzheimer's, but also for the acute prevention of neural damage due to stroke or injury . 33: SB-415286 In a separate program at Johnson&Johnson, research focused on bisindolylmaleimides with PKC activity [132, 133]. The maleimide H-bond was important for potency as methylation (36) abolished activity as a GSK3 inhibitor. Some selectivity was gained by varying the size of the macrocycle formed by tethering the indoles together. Where n = 2 or 3 (37), GSK3 IC50's were 22 nM and 26 nM respectively. While a smaller ring (n = 1) reduced potency, a larger ring size (n = 4) only doubled the IC50 to 51nM. In 37, bisindoles (A = B = CH) or mixed indole/azaindole (A = CH, B = N) compounds showed dual activity inhibiting both PKCγ (1.2-5.2 uM) and GSK3β (17-136 nM). Much greater selectivity toward GSK3β was gained from incorporating a nitrogen into both of the indole benzyl rings (38). Not only did the 7-azaindoles 38 impart selectivity for PKC (IC50 = >10uM), they were also much more selective against CDK1, CDK2 and VEGF-R. Computational modeling of these structures into the active stie of GSK3 helps to explainthe selectivity. One of the azaindoles makes a potential third Hbond with the Arg-141 in GSK3 which is unique to this ATP binding pocket. These 7-azaindoles exhibited good selectivity against a 50 kinase panel. Both optimal compounds (38) were able to stimulate GS activity in HEK293 cells. R O H N O O Cl Ph O N 34: SB-216763 O N A Cl N O B N N O 35: LY-294002 O O n 36: R = Me, A = B = CH, n = 2 37: R = H, A = B = CH, n = 2, 3 38: R = H, A = B = N, n = 2, 3 Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1121 5-ARYL-PYRAZOLOPYRIDAZINES AND PYRIDINES IC50 = 600nM and CDK5/p25 with an IC50 of 400nM. With the similarity between their ATP binding sites, it is not unexpected that these compounds also inhibited GSK3β at comparable concentrations, IC 50 = 1 uM. Only compound 44 was tested against GSK3β, and none of the other compounds was more potent against the CDK's than 44. Further work at GlaxoSmithKline identified pyridazine 39 (IC 50 = 250nM) as a lead in a new series of GSK3 inhibitors . Related compounds have been published in patent applications by GSK [135-137] and Vertex . The C-4 phenyl and N at the 6 position could be removed without significant loss of activity. The IC50 of these compounds could be increased considerably by introducing a small lipophilic amide on the C-3 amino group. Fluoro and chloro groups on the ortho position of the C-5 arene also lowered the IC50s to the 20nM range. Compounds 40 and 41 were the most potent of this series with IC50's of 5nM and 7nM respectively. Computational modeling helps to explain the SAR with the major H-bonding occurring from the amino-pyrazole of the compounds to GSK3's backbone Asp 133 and Val 135. In a kinase selectivity panel, excellent selecti-vity was observed except for CDK2 which has a closely related homology at the ATP binding site. Further refine-ments based on the Xray crystal structure-based design led to the highly potent (GSK3α IC 50 = 22nM) compound 42 which takes advantage of a H-bond to a structural water in the active site . The introduction of a dimethylamine solublizing group to the C-3 amide lead to vastly improved CDK2 selectivity. Compound 42 was nearly 700 fold more selective for GSK3 over CDK2 while maintaining it's excellent selectivity in the kinase panel. Interestingly, a series of 4, 7-dihydro-2H-pyrazolo[3, 4-b]pyridines (43) was filed in an application by Sanofi-Synthelabo, Fr./Mitsubishi-Tokyo Pharmaceuticals, Inc . These compounds while structurally comparable to 40 could conceivably undergo oxidative aromatization to produce 1Hpyrazolo[3, 4-b]pyridines. 1, 3, 4- AND 1, 2, 5-OXADIAZOLES Novo Nordisk has published two approaches to discovery of novel GSK3 inhibitors. Using a virtual screening approach, a novel class of 1, 3, 4-oxadiazoles was identified with IC50's below 1uM. The method described was derived from the CATS (Chemically Advanced Template Search) molecular descriptor, which is a technique used to compare the topological pattern of atoms and functional groups in a pharmacophore. Through iterative refinement, libraries of compounds made on solid-support defined the SAR and lead to the most potent compound 45, IC 50 = 390nM . In a second program, optimization was based on an HTS-derived screening hit. A series of potent 1, 2, 5-oxadiazoles was explored [143, 144] and medicinal chemistry quickly established the requirement for a para-substituted pyridine to ensure low IC 50s. This group was attached to the main scaffold through a hydrazide which was subsequently replaced with an amide bond. Later improvements included the substitution of a triazole for the amide which improved potency and could potentially increase solubility. Compound 46 was found to be highly potent (IC 50 = 280nM) and selective (~100 fold) for GSK3β over CDK2. Compound 46 and 2 other lead compounds were used to characterize the GSK3 activity of the series. All were proficient at stimulating glycogen synthase activity and βcatenin protein expression in CHO-IR cells. Furthermore these compounds were able to increase glycogen synthase activity in isolated rat soleus muscle. This series exhibited good selectivity against a panel of 31 kinases. CYCLIN-DEPENDENT KINASE INHIBITORS WITH GSK3 ACTIVITY A series of pyrazolo[3, 4-b]quinoxalines were developed as inhibitors of cyclin-dependent kinases, such as CDK1/ cyclin B and CDK5/p25 . These compounds would be expected to exhibit similar H-bond donor and acceptor interactions to the pyrazolopyridines (above). The most potent compound in this class 44 inhibited CDK1/cyclin B with an NON-ATP COMPETITIVE THIADIAZOLIDINONES GSK3 INHIBITORS A family of thiadiazolidinones has been described as having potential for treating Alzheimer's disease pathology. O O HN 4 5 N 6 F N 3 N2 N H N 41 40 N 39 N F N H N N H1 N 7 HN NH 2 NH O HN Cl NH 2 F F HN N N N H N 42 N N F NMe2 O N H N O 43 44 1122 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. These compounds show non-competitive inhibitor kinetics in relation to varied ATP or inhibitor concentrations in contrast to the ATP competitive comparator, Ro 318220 (1) [145, 146]. While the inhibition of GSK3β was modest (IC50's in the low uM range see 47 below, IC50 = 2uM), non-competitive inhibitors may show marked differences in activity between substrates and might also effect GSK3's ability to form protein-protein associations in complexes necessary to initiate tau phosphorylation or the Wnt pathway through presenilin 1. These compounds did show excellent selectivity with no detectable inhibition of CDK1/cyclin B, CK-II, PKA or PKC at 100uM. It will be interesting to determine if these thiadiazolidinones suppress tau hyperphosphorylation in whole cell studies. MISCELLANEOUS GSK3 INHIBITORS Several patent applications from Vertex have published describing small molecule GSK3 inhibitors. A recent application discloses a scaffold which incorporates a pyrimidine / pyridine moiety . Compound 50 was reported to have a Ki <100nM. Also disclosed were pyrazolone 51 , pyrazolamine derivatives 52 as inhibitors of GSK3, Aurora-2 and CDK-2 [151-158] and triazoles such as 53 with Ki's <100nM for GSK3β . Janssen Pharmaceutica has filed patent applications on several series of GSK3 inhibitor all based on substituted amino pyrimidines 54 , 55  and 56 . Sanofi-Synthelabo, Fr./Mitsubishi-Tokyo Pharmaceuticals, Inc  most recently published a patent application describing a rotationally restricted analog 57 of their earlier series 59. 2-pyrimidinyl- 6, 7, 8, 9- tetrahydropyrimido [1, 2-a] pyrimidin- 4-ones 57 and 7-pyrimidinyl- 2, 3-dihydroimidazo [1, 2-a]pyrimidin- 5(1H)- ones were specifically disclosed to be useful for the treatment of neurodegenerative diseases. In this case, the C-7 pyridinyl group was replaced with a pyrimidinyl group. The C-7 puridinyl 2, 3-dihydroimidazo analogues 58 were also specifically mentioned for their potential in the treatment of neurodegenerative diseases . The remainder of the published intellectual property consists of MUSCARINIC AGONISTS AS INHIBITORS OF GSK3 M1 muscarinic agonists are used as therapeutics to enhance cognition, but also have an affect on decreasing tau hyperphosphorylation in Alzheimer's disease . Two of this class of agonist AF102B (48) and AF150 (49) have been shown to reduce tau hyperphosphorylation through a GSK3 inhibition mechanism in cell studies . There may be a potential for synergy between classic GSK3 inhibitors along with muscarinic agonists or the non-competitive thiadiazolidinones for the treatment of Alzheimer's disease. S N N N N N N O N I N O N N N N N 45 O O F3 C 46 48: AF102B 47 49: AF150 50 N Me H N H N O N CN N N N NH 2 N N S S N O O NH H N HN N NH N HN N HN Me HN N F3 C N Cl O O O Me N N 53 52 51 N O O N N O N H 2N HN NH H N O NH O N N N HN 54 N 55 O- HN N+ O 56 N Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1123 the parent pyrimidones 59 claimed as potent GSK3β inhibitors for use as medicaments for prevention and/or treatment of diabetes, diabetic complications and neurodegenerative diseases or as immunopotentiators [165-171]. 5-a]-1, 3, 5-triazine derivatives 67 with activity as CDK and GSK3 inhibitors . These compounds were reported to inhibit the phosphorylation of histone H1 by CDK1/cyclin B1 in vitro. In a test for anti-proliferative activity against cancer cells, all compounds tested had activity against human pancreatic cancer cells Mia-PaCa2, and some had similar activity against human prostate cancer cells DU-145. Other applications claiming methods of treatment of conditions involving GSK3 include a method of antisense modulation of GSK3 α expression by Isis Pharmaceuticals, Inc.  and peptide inhibitors specific for GSK3 by an early researcher in the field, Prof. H. Eldar-Finkleman [181, 182]. Pfizer has filed patent applications on acylaminoimidazoles, for example 60, as inhibitors of CDK5, CDK2, and GSK3 . Also published are thiazole derivatives, for example 61, as inhibitors of CDKs and GSK3 for treating cancer, neurodegenerative diseases and conditions affected by dopamine neurotransmission. . Thiazoles were also the basis for two GSK3 patent applications by Novo Nordisk, although the thiazoles were more substituted than those of Pfizer, for example 62 and 63 [174, 175]. PHARMACOLOGY OF GSK3 INHIBITORS Other than the maleimides and pyrazolo[3, 4-c]pyridines, GSK has an application claiming triarylimidazoles for the treatment of diabetes . The compound 64 is reported to show an IC50 value in the range of 50 nM against GSK3β. Abnormal Glucose Metabolism in Type 2 Diabetes and Obesity Diabetes mellitus afflicts an estimated 17 million Americans and can lead to blindness, renal failure, limb amputations, and cardiovascular disease. The majority (>90%) of diabetics have type 2 diabetes which is characterized by resistance to insulin-mediated glucose metabolism and defective insulin secretion, see reviews [183-187]. Insulin resistance, which is also observed in obesity and metabolic syndrome (syndrome X) [188, 189], results in impaired Astrazeneca has filed patent applications on two series of structures for use in conditions involving the inhibition of GSK3, pyrazine-2-carboxamides 65  and anilinopyrimidines 66 . The Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.), Fr. has disclosed pyrazolo[1, N N R N N N N N O N N 58 R NH N N O 57 AcHN N R NH NH O N O 59 60 O O N O O Boc S S N N H N 61 O N H S NH 2 N N H 63 N NH 2 N F N N O S O2 O N H N N HN NH 2 N 62 N N N Ph N N 65 N HN Br 64 Br S O N N 66 N HN N N N 67 1124 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 glucose uptake and/or metabolism by the key regulatory peripheral tissues (muscle, adipose, and liver). One manifestation of impaired glucoregulation in these individuals is glucose intolerance which may progress to overt hyperglycemia. Mechanisms for impairment of glucose transport and glycogenesis in type 2 diabetics has been the subject of keen investigation for many years. A key concept of the diabetic phenotype, which is pertinent to studies of GSK3, is that muscle glycogen synthesis is the major pathway for glucose metabolism in the body and is defective in type 2 diabetics . While the primary basis for sub-optimal glycogenesis in type 2 diabetics may be linked to impaired glucose transport , it is apparent that rates of both glucose uptake and glycogen synthase (GS) can be limiting for glycogen accumulation . In the review of pharmacological data supporting the use of GSK3 inhibitors in the treatment of type 2 diabetes, we will focus on studies carried out at Chiron corporation using compounds from the pyridine / pyrimidide class. DISREGULATED GSK3 AND GS IN HUMAN TYPE 2 DIABETES Aside from the work described above with various models systems, a compelling set of studies from the laboratories of Drs. Shulman and Henry provided strong rationale for considering GSK3 as a target for novel therapeutic development. The investigators first showed that muscle tissue from type 2 diabetics, in comparison to normal tissue, had reduced glycogen content that correlated with decreased glycogen synthase activity and impaired GS responsiveness to insulin [192, 193]. A myocyte culture system was established which retained the GS characteristics of normal vs diabetic subjects and which exhibited appropriate coupling of glucose transport and glycogenesis [194, 195]. While the researchers had postulated early on that the reduced GS activity might be related to downregulation by kinase-mediated phosphorylation, the link was not solidified until a strong association with GSK3 was established . They showed that in obese, glucose-intolerant subjects, as well as type 2 diabetics, the reduced GS activity, and not protein level, correlated with an increase in total GSK3 activity that was related to increased α and β isoform levels. The link between GSK3 and attenuated GS activity was further supported by a series of reports using lithium . For example, it has been shown that lithium reduced hyperglycemia in a diabetic patient  and improved insulin sensitivity in diabetic rats . Lithium was also shown to increase hepatic glycogen synthesis in diabetic rats . More recently, inhibition of GSK3 by lithium was shown to increase both glucose transport and glycogen synthase activities in 3T3-L1 adipocytes . PHARMACOLOGICAL PROOF-OF-CONCEPT STRATEGY When discovery and optimization of GSK3 inhibitors was initiated at Chiron, there was no validation that potent and selective GSK3 inhibitors might represent effective therapeutics for type 2 diabetes. Hence, a proof-of-concept program was assembled involving both internal and external efforts. In vitro and in vivo models of glucose metabolism Wagman et al. were established internally and collaborations were secured with a number of laboratories as follows: Drs. Gary Cline and Gerald Shulman applied quantitative NMR methodology to analyze glucose metabolism in intact ZDF Fa/Fa rats, Drs. Robert Henry and Ted Ciaraldi investigated effects of inhibitors in human muscle cell cultures, Dr. Erik Henricksen studied muscle biopsies from normal or diabetic rats, Dr. Ormond MacDougald studied adipocyte regulation, and supplementary animal pharmacology studies were outsourced to GMI (aka Preclinomics, Indianapolis). All approaches proved productive and insightful and are described below. GSK3 INHIBITOR REGULATION OF GLUCOSE UPTAKE IN VITRO AND EX VIVO The potent and selective inhibitors CT 98014 and CT 98023 were tested in human skeletal muscle cell cultures after both acute and chronic exposure . The compounds rapidly increased glycogen synthase activity in normal and diabetic tissue of a magnitude greater than either lithium or insulin. Importantly, glucose transport was likewise upregulated, but in a manner that differed from that of insulin. Whereas insulin treatment yielded upregulated glucose transport over a time course of 1-12 hr, Chiron inhibitor treatment increased glucose uptake to a magnitude slightly greater than insulin (or lithium), but with delayed kinetics (i.e., a plateau extending from 24-96 hr of treatment). Interestingly, such upregulation was associated with a decrease in GSK3 protein levels and increase in IRS-1 protein levels - both of which would be desirable events should such a response be observed in the treatment of human diabetics. As a control, the inhibitor responses were shown to be specific for GSK3 inhibition as a structurallyrelated, but inactive compound had no effect. Similar studies were also performed with isolated muscles from lean and obese, diabetic ZDF fa/fa GMI rats, the latter of which are insulin-resistant and well-established as perhaps the premier animal model for type 2 diabetes [202, 203], reviewed in [204, 205]. Consistent with the human muscle cell cultures, ZDF rat muscle (following treatment ex vivo or after oral administration with ex vivo analysis) exhibited an increase in GS activity of a magnitude greater than insulin alone, and also showed an enhanced response upon exposure to an inhibitor/insulin combination [115, 201, 206]. Moreover, an upregulation of glucose transport in the presence of insulin following GSK3 inhibitor treatment was observed, although, the kinetics were not delayed in the same fashion as observed in the human muscle cultures. Notably, unlike the human muscle cell cultures wherein similar upregulation of glucose transport was apparent in normal and diabetic tissue, GSK3 inhibitor modulation of ZDF rat glucose uptake was stimulated in diabetic, but not lean rat muscle . Hence, the overall results with validated models in vitro and ex vivo supported the potential use of GSK3 inhibitors for the improvement of glucose metabolism in type 2 diabetes. ANIMAL PHARMACOLOGY APPROACH Given the novelty of GSK3 as a target for treating diabetes as well as the unique chemical structures identified and optimized, we reasoned that a broad range of animal models should be investigated for at least two reasons: First, Discovery and Development of GSK3 Inhibitors to provide confidence in the target and leads, and second, to guide clinical development through thorough characterization of the pharmacological profile. Hence, we utilized several mouse models of obesity and type 2 diabetes including ob/ob mice, db/db mice, and a “nongenetic” model of STZ/dietinduced type 2 diabetes [207, 208]. Such models were appropriate as they have been well validated for predictive value of other therapeutics. Furthermore, disregulation of a similar nature to that in the animals models was reported in diabetic humans. In particular, it was shown that C57BL/6J mice which are susceptible to diet-induced obesity and diabetes exhibited a two-fold increase in adipose GSK3 activity . Additionally, data presented at a Keystone symposium demonstrated that ob/ob mice exhibited an increase in muscle GSK3 activity which correlated with reduced GS activity and glucose intolerance . Chiron studies principally employed diabetic ZDF fa/fa rats. This model is well-established with defined and relevant biochemical and physiological endpoints [202-204, 207, 211]. A potential link to GSK3 stems from a report that the ZDF fatty rats exhibit downregulated IRS-1 and IRS-2 in liver and muscle . Additionally, Brozinick et al. have presented that obese, but not lean, ZDF fa/fa rats exhibited an increase in basal GSK3 activity in skeletal muscle . Additionally, we performed limited studies in obese, glucoseintolerant SHHF rats (GMI) and even lean STZ-treated rats; a model of type 1 diabetes with glucose intolerance. Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1125 6A Plasma exposure profile Plasma Insulin (ng/ml) Time Control 30 mg/kg -300 7.7 8.2 -10 7.3 7.8 25 9.4 8.4 6B Improved glucose tolerance with insulin conservation . To further validate both the concept and lead molecules, we performed a series of studies in obese, diabetic rhesus monkeys in collaboration with Dr. Barbara Hansen for the evaluation of pharmacokinetics and pharmacodynamics in primates . ORAL EFFICACY IN MOUSE MODELS OF TYPE 2 DIABETES Initial evaluations of the oral pharmacokinetics of CT 99021 in db/db mice showed oral bioavailability of ~25% with plasma concentrations at the tested dose in a predicted efficacious range based on in vitro potency (Fig. 6A). Impressive in vitro and ex vivo data led us to reason that it might be feasible to observe acute efficacy upon oral administration of the compounds. We also identified early on that the oral glucose tolerance test (oGTT), a common screen in animals and man for impaired glucose tolerance, showed responsiveness to GSK3 inhibitors after acute exposure. However, as the plasma half-life of CT 99021, and other similar molecules being screened, often ranged 1-2 hr in rodents we chose to dose twice prior to the oGTT (at 4.5 and 0.5 hr prior to glucose administration) to improve the sensitivity of the assay as well as to allow responsiveness to compounds of varying pharmacokinetics. As indicated in (Fig. 6B), oral administration of CT 99021 to db/db mice prior to an oral glucose challenge yielded a dose-related improvement in glucose disposal. At the higher dose, there was also a trend for reduced hyperglycemia prior to the glucose administration. As plasma insulin levels in the CT 99021-treated animals were similar, or even possibly conserved, relative to vehicle controls and as glucose levels in the urine were not elevated in treated rodents (not shown), it was concluded that there was an Fig. (6). Oral exposure and efficacy of CT 99021 administered to db/db mice. A) Plasma concentration vs time profile. 50 mg/kg CT 99021 formulated in 15% captisol administered once orally to 8-9 week old female db/db mice. Quantitation by LC/MS/MS. Results are mean + sem from 6 animals. B) Improved glucose tolerance with insulin conservation. Treatment with vehicle control (10% captisol) or indicated doses of CT 99021 at -4.5 and -0.5 hr relative to t = 0 oral glucose. Groups of 6-8 mice per treatment fasted for ~6 hr prior to a oGTT. SEM < 15%. Experimental methods as described in . improvement in whole body glucose disposal. Subcutaneous administration of another potent, but poorly orally bioavailable, compound (CT 98014) also exhibited improved glucose tolerance . The magnitude of efficacy in such studies 1126 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 could be quantified as the reduced glucose AUC for comparison between GSK3 inhibitors and reference therapeutics such as troglitazone, rosiglitazone, and metformin. The glucose AUC reduction of the 30 mg/kg regimen in the oGTT shown is of a magnitude similar to that observed following a metformin or troglitazone regimen. Notably, such efficacy by those therapeutics can not be observed within the first day of administration and typically require 12 weeks of daily dosing. Employing the same test compound and regimen, ob/ob mice were likewise tested in an oGTT model and, as indicated in (Fig. 7), improved glucose tolerance in an insulinconserving manner was observed. Within the age ranges tested, the ob/ob mice are less hyperglycemic and insulin 7A Improved glucose tolerance Wagman et al. resistant than the db/db mice and generally exhibited slightly greater efficacy upon comparison. Finally, we chose to extend the demonstration of acute oGTT efficacy to a mouse model not linked to a leptin or leptin receptor deficiency characteristic of ob/ob or db/db mice respectively . Consequently we applied an alternative model based on a high fat diet and intermittent streptozotocin treatment wherein animals progress to glucose intolerance and then overt diabetes  as is observed in man. Oral administration of CT 99021 twice prior to oral glucose challenge showed both a reduction in hyperglycemia and an impressive (53%) reduction in post-challenge glucose AUC (Fig. 8). The improved glucose disposal in the GSK3treated mice occurred without an increase in peripheral 7B Insulin conservation Fig. (7). Improved glucose tolerance with insulin sparing in ob/ob mice treated orally with CT 99021. A) Improved glucose tolerance. oGTT as described in figure 1 with 9-10 week old ob/ob mice. n = 6-8 animals/group, mean + sem. B) Insulin conservation. Plasma insulin levels (n = 6-8 animals/group, mean + sem). Fig. (8). Reduced hyperglycemia and improved glucose tolerance in nongenetic diabetic mice. Male C57BL/6J mice exposed to high fat diet and treated with STZ as described in . Vehicle control (15% captisol) or CT 99021 administered orally at 4.5 and 0.5 hr prior to oGTT. n = 8 animals/group, mean + sem. Discovery and Development of GSK3 Inhibitors Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1127 insulin levels (not shown) consistent with the ob/ob and db/db mouse oGTT results. Furthermore, the monitoring of blood glucose levels for an extended (up to 20 hr) period of time following vehicle control vs GSK3 inhibitor treatment in some of these models indicated that there was no compensatory rebound hyperglycemia in the treated animals (not shown). . We addressed the specificity of the GSK3 inhibitor oGTT response to insulin-resistant, glucose-intolerant animals rather than to normal animals using lean littermates of 10week-old obese, mildly diabetic animals. As seen in (Fig. 9A and 9B), the metabolically impaired animals showed normalization of their oral glucose tolerance following CT 99021 administration, whereas the lean littermates showed little change. [201, 206]. IMPROVED GLUCOSE TOLERANCE AND REDUCED HYPERGLYCEMIA IN RATS A more recently derived rat model of obesity and glucoseintolerance uses SHHF rats (Preclinomics/Charles River, Indianapolis IN) which also exhibit congestive heart failure. As the metabolic abnormality in SHHF rats is not linked to a leptin defect, it represented an additional element of concept validation. SHHF rats, like the nongenetic diabetic mice, showed an impressive reduction in hyperglycemia and improved glucose tolerance upon CT 99021 treatment (Fig. 10). We extended the pharmacological evaluation to appropriate rat models of obesity and diabetes. CT 99021 was assessed in 12-week-old ZDF fa/fa obese, diabetic rats for alteration of glucose disposal in a oGTT model as described above. At dose levels of 30-48 mg/kg administered orally, a significant improvement of glucose disposal accompanied by insulin sparing relative to controls was observed (Fig. 9A) 9A Obese ZDF rats Plasma Insulin (ng/ml + SEM) Time (min) Control 99021 -250 7.2+0.6 6.3+0.3 -45 6.9+0.3 5.3+0.3 25 8.5+0.7 4.8+0.2 60 7.8+0.8 5.2+0.5 9B Lean littermates Plasma Insulin (ng/ml + SEM) Time (min) Control 99021 -250 0.2+0.1 0.3+0.1 -45 0.2+0.1 0.3+0.1 25 0.4+0.1 0.6+0.1 60 0.3+0.1 0.4+0.1 Fig. (9). Differential responsiveness of obese vs lean ZDF rats to oral treatment with CT 99021. A) Obese ZDF rats. 10 week-old ZDF fa/fa rats fasted overnight and treated with vehicle control (captisol solution) or 30 mg/kg CT 99021 4.5 and 0.5 hr prior to oGTT. Methods as described above, n = 8/group, results are mean + sem. B) Lean littermates. Same as in A except age-matched lean littermates. 1128 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. 11A Reduced hyperglycemia Fig. (10). Improved glucose disposal in obese SHHF rats treated orally with CT 99021. Same study design as described in figure 4 except with 12 week-old obese and glucose-intolerant GMI SHHF rats. n = 8/group, results are mean + sem. Note reduced hyperglycemia after a single oral exposure prior to 2nd oral dose and subsequent oGTT. To better focus upon the regulation of glucose metabolism independent of glucose challenges, a series of studies were performed in ZDF rats following either acute or multiday sustained exposure to CT 99021. In acute studies, the GSK3 inhibitor was orally administered once to ZDF fa/fa rats with subsequent monitoring of plasma glucose and insulin levels (Fig. 11) . A dose-related and significant (at 30 mg/kg) reduction in hyperglycemia was observed which initiated within 60 min of administration and yielded a maximum reduction of >150 mg/dL. CT 99021-treated animals exhibited a trend for insulin lowering relative to the vehicle controls (Fig. 11B). Notably, the pharmacodynamics correlated well with the pharmacokinetics as the onset of glucose lowering coincided with plasma CT 99021 levels approaching maximal levels (Cmax) and with attenuation of the response coinciding with compound elimination (Fig. 1 and data not shown). In the longer-term studies, CT 99021 was continuously infused intravenously over a 4-day period at a total dose of ~92 mg/kg/d in order to ascertain the impact of high, sustained GSK3 inhibitor exposure on circulating glucose and insulin levels. Interestingly, a significant reduction in fasting hyperglycemia to nearly normoglycemic was observed without a significant change in plasma insulin (Table 2). 11B Insulin conservation Fig. (11). Oral CT 99021 reduces hyperglycemia with insulin conservation in ZDF Fa/Fa rats. A) Reduced hyperglycemia. 12 week old male ZDF fa/fa rats with food removal 3-4 hr prior to single oral administration of vehicle control (15% captisol) or indicated doses of CT 99021. Serial tail-snip bleeds from n = 8/group. B) Insulin conservation. Plasma insulin levels in same animals. Results are mean + sem. Table 2. QUANTITATIVE ANALYSIS OF GLUCOSE METABOLISM IN GSK3 INHIBITOR-TREATED ZDF RATS To provide insights into the mechanisms for glucoregulation by GSK3 inhibitor treatment in vivo 13C-NMR spectroscopy was applied. Glucose metabolism was studied in ZDF rats treated orally with CT 98023 prior to a 13C-oGTT and in hyperinsulinemic-euglycemic clamps following either acute (2-4 hr) or more chronic (20-24 hr) intravenous infusion of CT 99021. Regardless of the compound or regimen, the results suggested that hepatic glycogen formation was a primary disposal mechanism concomitant with reduced Effect of Sustained CT 99021 Exposure1 on ZDF Fa/Fa Rat Glycemia and Insulinemia Parameter Time Vehicle CT 99021 Glucose 2 (mg/dL) Pretreatment Day 4 188 + 18 181 + 20 223 + 18 116 + 6 Insulin 2 (ng/ml) Pretreatment Day 4 3.5 + 0.4 4.0 + 0.4 3.9 + 0.6 3.1 + 0.4 1 Intravenous prime (5 mg/kg) with continuous infusion at 64 ug/kg/min for 4 days to 12-13 week old ZDF Fa/Fa rats. 2 Plasma samples from overnight-fasted animals. endogenous glucose production . Specifically, in the oGTT studies, CT 98023 mediated a 41% reduced glucose Discovery and Development of GSK3 Inhibitors AUC with insulin sparing (26% reduced AUC) accompanied by a ~2-fold increase in hepatic glycogen formation and no significant increase in muscle glycogen content despite a ~3fold increase in GS activity. Likewise, in glucose clamp studies, a 2-3 fold increase in liver glycogen formation was observed, with a concomitant ~40% reduction in basal endogenous glucose output. Muscle GS activation was observed, although increases in glycogen formation were minimal. Hence, under these conditions, GSK3 inhibitor administration was shown for the first time to exhibit significant antidiabetic efficacy in vivo with a predominant effect on liver glucose metabolism. Glucose push in concert with activated GS likely accounted for increased glycogen synthesis . Reduced hepatic glucose output may likewise be direct as other investigators demonstrated downregulation of expression of the gluconeogenic enzymes (see above) . Acute ex vivo glucose uptake studies with obese, diabetic ZDF fa/fa muscle [115, 206] and human cells  would support the use of GSK3 inhibitors for the treatment of human diabetes. On the contrary the glucose clamp studies described above argue against human efficacy. This issue therefore remains an open question and awaits testing in man. Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1129 12A Improved glucose tolerance 12B Insulin sparing PHARMACOLOGICAL PROFILE IN RATS AND PRIMATES OF THE POTENT GSK3 INHIBITOR, CT 20026 The GSK3 inhibitor optimization cycle yielded a compound, CT 20026, with very good in vitro potency and selectivity. In initial animal screens it proved orally bioavailable and potent in vivo. Administration once via oral gavage to mildly diabetic ZDF rats 30 or 60 min prior to an oGTT yielded dose-related, significantly improved glucose disposal in an insulin-sparing manner (Fig. 12A, 12B), at a dose as low as 16 mg/kg. At a high dose (64 mg/kg), glucose tolerance was completely normalized. The compound showed similar potency and efficacy in mouse oGTT and reduced fasting hyperglycemia in ZDF rats and ob/ob mice. A number of studies were performed to evaluate the pharmacokinetics and pharmacodynamics of CT 20026 in rats and the best relationship arose from the correlation of plasma exposure (CT 20026 AUC) with oGTT efficacy (reduced glucose AUC). As depicted in (Fig. 13), a linear relationship (r2 = 0.71) was evident and higher exposure efficacy equaled that achievable by any therapeutic regimen including metformin (e.g., 100 mg/kg BID 1 week) or rosiglitazone (e.g., 5 mg/kg QD 2 weeks). Applying pharmacological relationships established in rodents, we advanced CT 20026 into acute efficacy studies in obese, diabetic rhesus monkeys. The physiology of these animals is well described the similarities of rhesus type 2 diabetes to that of man have been documented . We selected two mildly diabetic subjects and two markedly diabetic subjects. Preliminary pharmacokinetics demonstrated that the orally dosed compound showed a delayed plasma Tmax relative to rodents and an increased AUC per given dose as compared to rodents: a 5 mg/kg oral dose in the primates approximated the AUC of a 15 mg/kg oral dose in rats. We selected a relatively low dose (5 mg/kg) for assessing potential improvements in glucose tolerance. Due to the substantial variability in fasting glucose and insulin Fig. (12). Single administration oral efficacy of CT 20026 in ZDF Fa/Fa rats. A) Improved glucose tolerance. 10 week old male rats fasted overnight and administered a single oral dose of vehicle control (10% captisol) or increasing doses of CT 20026 30 min prior to oGTT. B) Insulin sparing. Plasma insulin levels monitored during oGTT study in same animals as in A. Results are from n = 8/group, mean + sem. levels between animals, we expressed the results as the percent change relative to pre-dose (and pre-oGTT) baselines. As shown in (Fig. 14), single oral administration of CT 20026 prior to an oGTT improved glucose disposal in an insulin-conserving manner relative to vehicle control responses in the same animals. Importantly, such efficacy was observed in the mildly diabetic primates, but not in the severely diabetic subjects which exhibited essentially controllevel glucose and insulin responses (data not shown). The differential magnitudes of glucose-lowering efficacy of GSK3-inhibitor treatment between mild and advanced diabetic animals – whether in acute or 1-2 week studies - has 1130 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. 14A Improved glucose tolerance Fig. (13). Oral PK/PD of CT 20026 in the ZDF fa/fa rat oGTT model. Results shown represent the summary of at least 6 separate studies in 9-11 week old, fasted male rats (n = 6-8 animals/group). Animals were treated with different oral dose levels of CT 20026 at 30-60 min prior to a standard oGTT. Plasma samples were analyzed for glucose and CT 20026 levels in order to compare the pharmacokinetics (PK) and pharmacodynamics (PD, e.g., glucoselowering). The zone of maximum achievable efficacy pertains to the range of maximal reduced glucose AUC (trapezoidal) that can be obtained in this animal model with any treatment. (GSK3 inhibitor or optimal treatment regimens with troglitazone, rosiglitazone, or metformin). 14B Insulin conservation also been observed in our mouse and rat models and may be a general phenomenon. Whether the basis is simply related to greater insulin resistance in the advanced diabetics or whether there is a quantal limit of glucose “disposal” which is proportionally less in the markedly hyperglycemic subjects is currently unclear. CHRONIC CT 20026 ADMINISTRATION METABOLIC REGULATION AND To ascertain the therapeutic potential of GSK3 inhibitor treatment for type 2 diabetes we evaluated long-term efficacy in ZDF fa/fa rats. We began treating animals at 8-9 weeks for 30 days with CT 20026 at 16 mg/kg each morning and evening. The regimen was well-tolerated and, as indicated in (Fig. 15), diabetic progression (as measured by changes in HbA1c and insulinemia) was prevented in the treated animals. While the terminal HbA1c was significantly reduced in the CT 20026-treated animals relative to the vehicle controls, it is worth noting that treatment with 5 mg/kg rosiglitazone over a similar period lowers glucose more profoundly (data not shown). Also, in separate studies with older, more hyperglycemic and insulin-resistant ZDF rats, the same regimen of CT 20026 failed to significantly lower HbA1c levels (data not shown). Paradoxically, insulin levels in the CT 20026-treated animals trended higher than the vehicle controls over the last 2 weeks of exposure. However, this insulin profile is consistent with reduced diabetic progression by GSK3inhibitor treatment as it is well-established that ZDF fa/fa Fig. (14). Oral efficacy of CT 20026 in mildly diabetic obese rhesus monkeys. A) Improved glucose tolerance. Two obese male rhesus rhesus monkeys with fasting plasma glucose levels 101 and 129 mg/dL (normoglycemic rhesus = 65-70 mg/dL) were given a single oral gavage of vehicle control (5% HPBCD solution) or 5 mg/kg CT 20026 1-2hr prior to an oGTT. Results are the average of the two animals from the pre-dose baseline level. B) Insulin conservation. Results are the average plasma insulin levels of the two animals from the pre-dose baseline level (139 and 58 ng/ml, respectively). rats, like humans, become insulinopenic with advancing diabetes. Additional support for such an interpretation of insulin dynamics comes from the observation that chronic rosiglitazone treatment of ZDF fa/fa rats prevents the loss in beta-cell mass thereby sustaining insulin levels . Hence, GSK3-inhibitor treatment prevents both the increase in hyperglycemia and reduction of insulinemia observed in developing diabetes in ZDF rats. Additional endpoints were followed in the chronic ZDF rat study. At termination, there was no significant change in lipid or cholesterol profiles nor other clinical chemistries relative to the vehicle controls. Muscle glycogen content Discovery and Development of GSK3 Inhibitors increased slightly and liver glycogen content was significantly increased by 49%. This is a physiologically meaningful increased and likely to be beneficial. Finally, there was a trend for increased body weight in the CT 20026-treated rats (Fig. 15C) that is also consistent with improved metabolism. Similar increases in weight gain can be observed in chronic rodent (and human) studies with other effective diabetes therapeutics. 15A HbA1c change Current Pharmaceutical Design, 2004, Vol. 10, No. 10 1131 ALTERNATIVE POTENTIAL EFFECTS OF GSK3INHIBITOR TREATMENT As GSK3 may participate in a number of regulatory processes (for review see: [9, 10, 27, 76]), it is possible that sustained treatment with a GSK3 inhibitor may have therapeutic utility in other diseases, as well as potentially undesirable side effects. Other therapeutic utilities may include the treatment of neurodegenerative diseases like Alzheimers or ischemia-reperfusion injury . Alternatively, potential upregulation of β-catenin may predispose recipients to certain types of cancer (for review see: [9, 10, 27, 76]). We have chosen to focus upon potential applications of GSK3 inhibitors for metabolic disorders in this review. Nonetheless, we note that chronic lithium treatment has not been associated with tumorigenesis in man. POSITIONING GSK3 INHIBITORS IN THE CLINIC AS METABOLIC DISEASE THERAPEUTICS 15B Plasma insulin 15C Body weight change As presented here, GSK3 inhibitors clearly have potential as novel therapeutics for the treatment of certain metabolic diseases. The pharmacological profile and non-clinical validation supports their use as a insulin-mimetics or insulinsensitizers. Despite the advances in diabetes therapeutics in recent years, there is still a tremendous unmet medical need – particularly for agents with novel modes of action. While GSK3 inhibitors may act alone, they would likely be beneficial in combination with insulin or other “sensitizers” such as thiazolidinediones or metformin. In fact, combination studies in rodent models, enhanced glucose lowering efficacy observed in the combination groups (data not shown). A strategic requirement for potential clinical development is identification of the patient type likely to be most responsive to the drug candidate. As described above in multiple animal species and in either acute or chronic settings, the glucose lowering efficacy of the Chiron GSK3 inhibitors was most evident in glucose-intolerant animals ranging from the pre-diabetic to moderately diabetic stage. Aside from regulation of glucose metabolism, GSK3 inhibitors may also have direct effects on obesity via regulation of adipocyte function [10, 218, 219]. Accordingly, we would propose that GSK3 inhibitor treatment is best suited for pre-diabetic obese and/or metabolic syndrome patients and early-stage type 2 diabetics. Moreover, given the efficacy of GSK3 inhibitors in improving post-prandial glucose disposal and potentially reducing hepatic glucose output, we anticipate that an optimal dosing regimen would provide effects throughout the day and night. ACKNOWLEDGEMENTS Fig. (15). Efficacy of oral CT 20026 administered for 1 month to ZDF fa/fa rats. Treatment of 8 animals/group beginning at 8-9 weeks of age with control (0.5% CMC suspension) or 16 mg/kg CT 20026 every morning and evening for 30 days. A) HbA1c change. B) Plasma insulin. C) Body weight change. Results are mean + sem. We are grateful for the contributions from our: Collaborators: R. Henry, T. Ciaraldi, G. Shulman, G. Cline, B. Hansen, E. Henricksen. Advisors: G. Reaven, R. Sherwin. Colleagues: S. Harrison, D. Ring, R. Johnson, E. Tozzo, L. Seely, J. Plattner, M. Desai, I. Samuels, G. Dollinger, K. Wang. Special thanks to Dr. Gary Cline, Yale, for critical review of the pharmacology section of this manuscript, and to Dr. Steven Harrison, for critical review of the completed manuscript and for useful discussions. 1132 Current Pharmaceutical Design, 2004, Vol. 10, No. 10 Wagman et al. 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