In type 2 diabetes, the liver produces excessive amounts of glucose through the gluconeogenesis (GNG) pathway and consequently is partly responsible for the elevated glucose levels characteristic of the disease. In an effort to find safe and efficacious GNG inhibitors, we targeted the AMP binding site of fructose 1,6-bisphosphatase (FBPase). The hydrophilic nature of AMP binding sites and their widespread use for allosteric regulation of enzymes in metabolic pathways has historically made discovery of AMP mimetics suitable for drug development difficult. By using a structure-based drug design strategy, we discovered a series of compounds that mimic AMP but bear little structural resemblance. The lead compound, MB05032, exhibited high potency and specificity for human FBPase. Oral delivery of MB05032 was achieved by using the bisamidate prodrug MB06322 (CS-917), which is converted to MB05032 in two steps through the action of an esterase and a phosphoramidase. MB06322 inhibited glucose production from a variety of GNG substrates in rat hepatocytes and from bicarbonate in male Zucker diabetic fatty rats. Analysis of liver GNG pathway intermediates confirmed FBPase as the site of action. Oral administration of MB06322 to Zucker diabetic fatty rats led to a dose-dependent decrease in plasma glucose levels independent of insulin levels and nutritional status. Glucose lowering occurred without signs of hypoglycemia or significant elevations in plasma lactate or triglyceride levels. The findings suggest that potent and specific FBPase inhibitors represent a drug class with potential to treat type 2 diabetes through inhibition of GNG.endogenous glucose production ͉ AMP mimetic ͉ structure-based drug design ͉ phosphonate prodrug ͉ antihyperglycemic T he liver (1, 2) and, to a lesser extent, the kidneys (3) are the primary organs responsible for endogenous glucose production (EGP). In type 2 diabetes mellitus (T2DM), excessive EGP in the fasted state and fed state contributes to the chronic elevation of blood glucose levels found in patients with advanced (4, 5) and mild diabetes (6, 7). Moreover, fasting plasma glucose levels correlate with EGP rates in patients with fasting plasma glucose Ͼ180 mg͞dl (10 mM) (8) and possibly in patients with lower levels (6, 7). Studies using 13 C NMR spectroscopy (9) as well as more recent studies using deuterated water (7, 10) attribute the excessive EGP in T2DM patients to increased flux through the gluconeogenesis (GNG) pathway.Efforts over the past 40 years to discover inhibitors of GNG produced few safe and effective drug candidates (11). Metformin, the only marketed drug that acts, at least partially, through inhibition of GNG (12), inhibits GNG indirectly and only 33-36% at the rarely prescribed maximal human dose (13). Direct GNG inhibitors show more pronounced glucose lowering in animals but not without eliciting safety-related concerns. Hypoglycemia, lactic acidosis, and hypertriglyceridemia are the principle safety risks associated with GNG inhibition as highlighted in studie...

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models. [Mol Cancer Ther 2009;8(4):921-9] IntroductionHeat shock protein 90 (Hsp90) is a widely expressed molecular chaperone that functions in the maturation and stabilization of cellular proteins (1-3). Hsp90, in complex with other cochaperone proteins, catalyzes the conformational changes of client proteins via its ATPase activity (4). The activity of Hsp90 maintains a variety of client proteins in their active conformation (5). Hsp90 also plays an important role in the regulation of several key oncogenic signaling proteins (6-8) and steroid receptors (9). Mutated proteins are particularly dependent on Hsp90 for the maintenance of the active conformation (2, 3).Ansamycin drugs such as geldanamycin bind in the ATPbinding site in the NH 2 terminus of Hsp90 (6, 10). This binding inhibits the chaperone activity of Hsp90 and results in proteasomal degradation of the client proteins (5, 11-13). Because tumor cells rely on the activity of client proteins for cell proliferation and survival, drug-induced client protein degradation leads to cytostasis and/or selective cell killing of tumor cell in vitro and in vivo (14-16).The semisynthetic Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) is currently in clinical trials for cancer (17)(18)(19). However, 17-AAG is expensive to prepare and difficult to formulate. The problematic nature of the formulations may well contribute to the dose-limiting toxicity observed with this compound. 17-AAG is also susceptible to metabolism by NQO1/DT-diaphorase enzymes (20) and to efflux by P-glycoprotein (21). The identification of a synthetic Hsp90 inhibitor would be of great therapeutic interest as it would circumvent t...

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

AMP binding sites are commonly used by nature for allosteric regulation of enzymes controlling the production and metabolism of carbohydrates and lipids. Since many of these enzymes represent potential drug targets for metabolic diseases, efforts were initiated to discover AMP mimics that bind to AMP-binding sites with high affinity and high enzyme specificity. Herein we report the structure-guided design of potent fructose 1,6-bisphosphatase (FBPase) inhibitors that interact with the AMP binding site on FBPase despite their structural dissimilarity to AMP. Molecular modeling, free-energy perturbation calculations, X-ray crystallography, and enzyme kinetic data guided our redesign of AMP, which began by replacing the 5'-phosphate with a phosphonic acid attached to C8 of the adenine base via a 3-atom spacer. Additional binding affinity was gained by replacing the ribose with an alkyl group that formed van der Waals interactions with a hydrophobic region within the AMP binding site and by replacing the purine nitrogens N1 and N3 with carbons to minimize desolvation energy expenditures. The resulting benzimidazole phosphonic acid, 16, inhibited human FBPase (IC50 = 90 nM) 11-fold more potently than AMP and exhibited high specificity for the AMP binding site on FBPase. 16 also inhibited FBPase in primary rat hepatocytes and correspondingly resulted in concentration-dependent inhibition of the gluconeogenesis pathway. Accordingly, these results suggest that the AMP site of FBPase may represent a potential drug target for reducing the excessive glucose produced by the gluconeogenesis pathway in patients with type 2 diabetes.

Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.

Excessive glucose production by the liver coupled with decreased glucose uptake and metabolism by muscle, fat, and liver results in chronically elevated blood glucose levels in patients with type 2 diabetes. Efforts to treat diabetes by reducing glucose production have largely focused on the gluconeogenesis pathway and rate-limiting enzymes within this pathway such as fructose-1,6-bisphosphatase (FBPase). The first potent FBPase inhibitors were identified using a structure-guided drug design strategy (Erion, M. D.; et al. J. Am. Chem. Soc. 2007, 129, 15480-15490) but proved difficult to deliver orally. Herein, we report the synthesis and characterization of a series of orally bioavailable FBPase inhibitors identified following the combined discoveries of a low molecular weight inhibitor series with increased potency and a phosphonate prodrug class suitable for their oral delivery. The lead inhibitor, 10A, was designed with the aid of X-ray crystallography and molecular modeling to bind to the allosteric AMP binding site of FBPase. High potency (IC50 = 16 nM) and FBPase specificity were achieved by linking a 2-aminothiazole with a phosphonic acid. Free-energy perturbation calculations provided insight into the factors that contributed to the high binding affinity. 10A and standard phosphonate prodrugs of 10A exhibited poor oral bioavailability (0.2-11%). Improved oral bioavailability (22-47%) was achieved using phosphonate diamides that convert to the corresponding phosphonic acid by sequential action of an esterase and a phosphoramidase. Oral administration of the lead prodrug, MB06322 (30, CS-917), to Zucker Diabetic Fatty rats led to dose-dependent inhibition of gluconeogenesis and endogenous glucose production and consequently to significant blood glucose reduction.

Therapeutic development for Alzheimer's disease has largely focused on the removal of beta amyloid because of its suggested role in the primary agent in initiating the disease process. However, with the recent discovery of mutations that result as pathologic buildup of tau in the absence of amyloid pathology, tau is beginning to be recognized as a potential target for drug discovery. We have developed a high-throughput drug screening method that allows for direct intracellular quantitation of tau protein species, enabling the fast, reliable detection of these changes. We have identified a family of small, blood brain barrier penetrant heat shock protein 90 inhibitors that significantly reduce tau protein levels in vitro. Western blot analysis demonstrated a clear inverse correlation between the tau levels and the increase in HSP27, HSP40 and HSP90. Modifications to this assay will further allow the specific analysis of pathologically relevant species. Using this assay, we have demonstrated that a class of HSP90 inhibitors is able to significantly lower intracellular tau levels most likely through induction of a heat shock response.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.