We investigated the role of glycogen synthase kinase-3 (GSK-3), which is inactivated by AKT, for its role in the regulation of apoptosis. Upon IL-3 withdrawal, protein levels of MCL-1 decreased but were sustained by pharmacological inhibition of GSK-3, which prevented cytochrome c release and apoptosis. MCL-1 was phosphorylated by GSK-3 at a conserved GSK-3 phosphorylation site (S159). S159 phosphorylation of MCL-1 was induced by IL-3 withdrawal or PI3K inhibition and prevented by AKT or inhibition of GSK-3, and it led to increased ubiquitinylation and degradation of MCL-1. A phosphorylation-site mutant (MCL-1(S159A)), expressed in IL-3-dependent cells, showed enhanced stability upon IL-3 withdrawal and conferred increased protection from apoptosis compared to wild-type MCL-1. The results demonstrate that the control of MCL-1 stability by GSK-3 is an important mechanism for the regulation of apoptosis by growth factors, PI3K, and AKT.

Insulin resistance plays a central role in the development of type 2 diabetes, but the precise defects in insulin action remain to be elucidated. Glycogen synthase kinase 3 (GSK-3) can negatively regulate several aspects of insulin signaling, and elevated levels of GSK-3 have been reported in skeletal muscle from diabetic rodents and humans. A limited amount of information is available regarding the utility of highly selective inhibitors of GSK-3 for the modification of insulin action under conditions of insulin resistance. In the present investigation, we describe novel substituted aminopyrimidine derivatives that inhibit human GSK-3 potently (K i < 10 nmol/l) with at least 500-fold selectivity against 20 other protein kinases. These low molecular weight compounds activated glycogen synthase at ϳ100 nmol/l in cultured CHO cells transfected with the insulin receptor and in primary hepatocytes isolated from Sprague-Dawley rats, and at 500 nmol/l in isolated type 1 skeletal muscle of both lean Zucker and ZDF rats. It is interesting that these GSK-3 inhibitors enhanced insulin-stimulated glucose transport in type 1 skeletal muscle from the insulin-resistant ZDF rats but not from insulin-sensitive lean Zucker rats. Single oral or subcutaneous doses of the inhibitors (30 -48 mg/kg) rapidly lowered blood glucose levels and improved glucose disposal after oral or intravenous glucose challenges in ZDF rats and db/db mice, without causing hypoglycemia or markedly elevating insulin. Collectively, our results suggest that these selective GSK-3 inhibitors may be useful as acute-acting therapeutics for the treatment of the insulin resistance of type 2 diabetes. Diabetes 52: 588 -595, 2003

T he phosphoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival, intracellular trafficking, and membrane ruffling. 1 PI3K's can be categorized into class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3K's are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110R, β, and δ. A single class IB PI3K, activated by GPCRs, consists of only one member: a p110γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3K's is phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2), which upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3 (PIP3) serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. 5À10 We have previously reported on a series of 6-hydroxyphenyl-2-morpholino pyrimidines, 11 as potent pan class I PI3K inhibitors that exhibit high selectivity toward protein kinases (serine/threonine and tyrosine kinases). We have further reported on non-phenol containing heterocyclic, morpholino pyrimidines 12 such as compound 1 which demonstrate in vivo PI3K pathway modulation and modest tumor growth inhibition. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of class I PI3Ks that exhibit potency and pharmacokinetic properties which allow for maximal pathway modulation in vivo and have druglike properties suitable for clinical development. These efforts culminated in the identification of 15, NVP-BKM120.Aminopyrimidine 1 and analogues such as 3 (Figure 1) exhibit low or sub-nanomolar biochemical potency and sub-micromolar cellular potency against PI3KR. Even with high rodent CL values, such analogues can demonstrate PI3K pathway modulation in mouse xenograft models. 12 During our exploration of the C 6 position, it was noted that C 6 aminopyridine analogue 4, while being less potent than 3 against PI3KR (>10Â potency loss), exhibited a markedly reduced (>9Â) rat CL value, increased %F, and increased oral AUC. Thus, superior pharmacokinetic properties were achievable within this scaffold and the challenge remaining was to retain this kind of pharmacokinetic profile while optimizing all the other attributes (potency, solubility, permeability, safety) necessary for advancement. To address this challenge, ...

Aims/hypothesis: Treatment with glucocorticoids, especially at high doses, induces insulin resistance. The aims of the present study were to identify the potential defects in insulin signalling that contribute to dexamethasone-induced insulin resistance in skeletal muscles, and to investigate whether the glycogen synthase-3 (GSK-3) inhibitor CHIR-637 could restore insulin-stimulated glucose metabolism. Materials and methods: Skeletal muscles were made insulin-resistant by treating male Wistar rats with dexamethasone, a glucocorticoid analogue, for 12 days. Insulin-stimulated glucose uptake, glycogen synthesis and insulin signalling were studied in skeletal muscles in vitro. Results: Dexamethasone treatment decreased the ability of insulin to stimulate glucose uptake, glycogen synthesis and glycogen synthase fractional activity. In addition, the dephosphorylation of glycogen synthase by insulin was blocked. These defects were paralleled by reduced insulin-stimulated protein kinase B (PKB) and GSK-3 phosphorylation. While expression of PKB, GSK-3 and glycogen synthase was not reduced by dexamethasone treatment, expression of the p85α subunit of phosphatidylinositol 3-kinase (PI 3-kinase) was increased. Inhibition of GSK-3 by CHIR-637 increased glycogen synthase fractional activity in soleus muscle from normal and dexamethasone-treated rats, although the effect was more pronounced in control rats. CHIR-637 did not improve insulin-stimulated glucose uptake in muscles from dexamethasone-treated rats. Conclusions/interpretation: We demonstrated that chronic dexamethasone treatment impairs insulin-stimulated PKB and GSK-3 phosphorylation, which may contribute to insulin resistance in skeletal muscles. Acute pharmacological inhibition of GSK-3 activated glycogen synthase in muscles from dexamethasone-treated rats, but GSK-3 inhibition did not restore insulinstimulated glucose uptake.

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 GSK3beta, 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.

Decades of preclinical and natural history studies have highlighted the potential of fatty acid synthase (FASN) as a bona fide drug target for oncology. This review will highlight the foundational concepts upon which this perspective is built. Published studies have shown that high levels of FASN in patient tumor tissues are present at later stages of disease and this overexpression predicts poor prognosis. Preclinical studies have shown that experimental overexpression of FASN in previously normal cells leads to changes that are critical for establishing a tumor phenotype. Once the tumor phenotype is established, FASN elicits several changes to the tumor cell and becomes intertwined with its survival. The product of FASN, palmitate, changes the biophysical nature of the tumor cell membrane; membrane microdomains enable the efficient assembly of signaling complexes required for continued tumor cell proliferation and survival. Membranes densely packed with phospholipids containing saturated fatty acids become resistant to the action of other chemotherapeutic agents. Inhibiting FASN leads to tumor cell death while sparing normal cells, which do not have the dependence of this enzyme for normal functions, and restores membrane architecture to more normal properties thereby resensitizing tumors to killing by chemotherapies. One compound has recently reached clinical studies in solid tumor patients and highlights the need for continued evaluation of the role of FASN in tumor cell biology. Significant advances have been made and much remains to be done to optimally apply this class of pharmacological agents for the treatment of specific cancers.

Concurrent with the spread of the western lifestyle, the prevalence of all types of diabetes is on the rise in the world's population. The number of diabetics is increasing by 4-5% per year with an estimated 40-45% of individual's over the age of 65 years having either type II diabetes or impaired glucose tolerance. Since the signs of diabetes are not immediately obvious, diagnosis can be preceded by an extended period of impaired glucose tolerance resulting in the prevalence of beta-cell dysfunction and macrovascular complications. In addition to increased medical vigilance, diabetes is being combatted through aggressive treatment directed at lowering circulating blood glucose and inhibiting postprandial hyperglycemic spikes. Current strategies to treat diabetes include reducing insulin resistance using glitazones, supplementing insulin supplies with exogenous insulin, increasing endogenous insulin production with sulfonylureas and meglitinides, reducing hepatic glucose production through biguanides, and limiting postprandial glucose absorption with alpha-glucosidase inhibitors. In all of these areas, new generations of small molecules are being investigated which exhibit improved efficacy and safety profiles. Promising biological targets are also emerging such as (1) insulin sensitizers including protein tyrosine phosphatase-1B (PTP-1B) and glycogen synthase kinase 3 (GSK3), (2) inhibitors of gluconeogenesis like pyruvate dehydrogenase kinase (PDH) inhibitors, (3) lipolysis inhibitors, (4) fat oxidation including carnitine palmitoyltransferase (CPT) I and II inhibitors, and (5) energy expenditure by means of beta 3-adrenoceptor agonists. Also important are alternative routes of glucose disposal such as Na+-glucose cotransporter (SGLT) inhibitors, combination therapies, and the treatment of diabetic complications (eg. retinopathy, nephropathy, and neuropathy). With may new opportunities for drug discovery, the prospects are excellent for development of innovative therapies to effectively manage diabetes and prevent its long term complications. This review highlights recent (1997-2000) advances in diabetes therapy and research with an emphasis on small molecule drug design (275 references).

Enzymes belonging to the phospholipase C (PLC) family hydrolyze the phosphodiester bond of phospholipids to give a diacylglycerol and a phosphorylated head group. "he bacterial phospholipase C from Bacillus cereus (PLCB,) has been studied extensively, and there is a wealth of information regarding those structural features that are important for substrate activity. In contrast, there is virtually no data available regarding structure-activity relationships for inhibitors of this enzyme. To address this shortcoming, a series of optically pure analogues of 1,2-dihexanoyl-sn-glycero-3phosphocholine (2) containing different replacements of the phosphate group were first synthesized including the phosphoramidates 4 and 8, the phosphonate 5, the (difluoromethy1ene)phosphonate 6, the thiophosphate 7, the diastereomeric phosphorothioates 9 and 10, and the phosphorodithioate 11. Each of these phosphatidylcholine derivatives was tested for inhibitor or substrate activity with PLCB, using the water-soluble phosphatidylcholine 2 as the monomeric substrate. The measurements were conducted below the critical micellar concentrations of both 2 and the inhibitor. Of the analogues, only 7 and 9 underwent observable enzymatic hydrolysis under the assay conditions used. The kcat of the (Sp)-phosphorothioate 9 was approximately one-fifth that of 2, and when compared to 2, 7 was hydrolyzed only very slowly by the enzyme. Kinetic studies indicated that the phospholipid analogues tested were competitive inhibitors with increasing K{s as follows: 7 x 11 x 10 < 4 x 8 5 x 6.(2) For reviews of the roles of DAG and IPS as second messengers, see: (a) Berridge, M. J. Biochem. (5)(a) Larroders, P.; Cornet, M. E.; Diaz-Meco, M. T.; Lopez-Barahona, M.; Diaz-Laviada, I.; Guddal, P. H.; Johnsen, T.; Moscat, J. Cell 1990,61,1113. (b) Garcia de Herreros, A.; Dominguez, I.; Diaz-Meco, M. T.; Graziani, G.; Cornet, M. E.; Guddal, P. H.; Johnsen, T.; Moscat, J. J. Biol. Chem. 1991,266, 6825. 0022-3263/94/1959-4821$04.50/0 phosphatidylcholine (PC). Recent reports have indicated that hydrolysis of phosphatidylcholine by both a phosphatidylcholine-specific PLC (PC-PLC) and phospholipase D (PLD) may contribute to the production of the second phase of DAG in the stimulated cell. The hydrolysis of PC by PC-PLC provides DAG directly, whereas hydrolysis by PLD provides phosphatidic acid that is further processed by phosphatidic acid phosphohydrolase to yield DAG.Recent experiments suggest that the PLC from B a d -Zus cereus (PLCB,) may be a useful model for mammalian PC-PLCS.~-~ This bacterial PLC has been well charact e r i~e d ,~ and its high-resolution (1.5 A) X-ray structure has recently been disclosed.1° Owing to these considerations coupled with the ready availability of PLCB,, we embarked upon an investigation to elucidate the mechanism of action of this enzyme to gain insights into the mechanistic details of the hydrolysis of the phosphodiester bond by enzymes of the PLC family. The first phase of this undertaking necessarily involved the design, prepa...