Srinivas Rao Kasibhatla

BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90

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]

MB06322 (CS-917): A potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2 diabetes.

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.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity

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.

Orally Active Purine-Based Inhibitors of Heat Shock Protein 90

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.

Development of a high throughput drug screening assay for the detection of changes in tau levels - Proof of concept with HSP90 inhibitors

Therapeutic development for Alzheimers 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.

Discovery of Phosphonic Diamide Prodrugs and Their Use for the Oral Delivery of a Series of Fructose 1,6-Bisphosphatase Inhibitors

Like most phosphonic acids, the recently discovered potent and selective thiazole phosphonic acid inhibitors of fructose 1,6-bisphosphatase (FBPase) exhibited low oral bioavailability (OBAV) and therefore required a prodrug to achieve oral efficacy. Syntheses of known phosphonate prodrugs did not afford the desired OBAV; hence, a new class of prodrugs was sought. Phosphonic diamides derived from amino acid esters were discovered as viable prodrugs, which met our preset goals: excellent aqueous stability over a wide pH range, benign byproducts (amino acids and low molecular weight alcohols), and most importantly good OBAV leading to robust oral glucose lowering effects. These desirable properties of phosphonic diamides represent significant improvements over existing prodrug classes. Optimization of the diamide prodrugs of phosphonic acid 2a (MB05032) led to the identification of diamide 8 (MB06322), the first reported orally efficacious FBPase inhibitor.

EC144 Is a Potent Inhibitor of the Heat Shock Protein 90.

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.