Brain-permeable small-molecule inhibitors of Hsp90 prevent alpha-synuclein oligomer formation and rescue alpha-synuclein-induced toxicity.

Aggregation of alpha-synuclein (alphasyn) is a hallmark of sporadic and familial Parkinson's disease (PD) and dementia with Lewy bodies. Lewy bodies contain alphasyn and several heat shock proteins (Hsp), a family of molecular chaperones up-regulated by the cell under stress. We have previously shown that direct expression of Hsp70 and pharmacological up-regulation of Hsp70 by geldanamycin, an Hsp90 inhibitor, are protective against alphasyn-induced toxicity and prevent aggregation in culture. Here, we use a novel protein complementation assay to screen a series of small-molecule Hsp90 inhibitors for their ability to prevent alphasyn oligomerization and rescue toxicity. By use of this assay, we found that several compounds prevented alphasyn oligomerization as measured by decreased luciferase activity, led to a reduction in high-molecular-mass oligomeric alphasyn, and protected against alphasyn cytotoxicity. A lead compound, SNX-0723 (2-fluoro-6-[(3S)-tetrahydrofuran-3-ylamino]-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide) was determined to have an EC(50) for inhibition of alphasyn oligomerization of approximately 48 nM and was able to rescue alphasyn-induced toxicity. In vivo assessment of SNX-0723 showed significant brain concentrations along with induction of brain Hsp70. With a low EC(50), brain permeability, and oral availability, these novel inhibitors represent an exciting new therapeutic strategy for PD.

Targeting of multiple signaling pathways by the Hsp90 inhibitor SNX-2112 in EGFR resistance models as a single agent or in combination with erlotinib.

Inhibition of Hsp90 has emerged as a therapeutic strategy to target NSCLC subtypes, which are refractory to epidermal growth factor receptor (EGFR) inhibitor-based treatment. We report on a novel small molecule inhibitor of Hsp90, SNX-2112, and an orally bioavailable prodrug analog, SNX-5422. In cellular models of wild-type or mutant EGFR (L858R and T790M mutations), SNX-2112 alone and in combination with erlotinib inhibited EGF activation of pAKT(473) and pSTAT3(705). pERK1/2 and pS6 were also potently inhibited by similar treatments. SNX-2112 reduced EGF cross-talk and activation of the c-Met receptor by causing c-Met degradation. In NCI-H1975 xenograft models, SNX-5422 showed activity as a single agent and in combination with erlotinib resulted in prolonged animal survival at reduced compound concentrations relative to either compound alone. These results support the advanced evaluation of SNX-5422 as a treatment for non-small cell lung cancer (NSCLC), especially in cases where the cancer is driven by c-Met amplification or mutated EGFR forms that are resistant to EGFR inhibitors.

SNX2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against HER kinase-dependent cancers.

PURPOSE: The heat shock protein 90 (Hsp90) chaperone plays an important role in transformation by regulating the conformational maturation and stability of oncogenic kinases and transcription factors. Ansamycins, such as 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), inhibit Hsp90 function; induce the degradation of Hsp90 client proteins such as HER2, and have shown activity in early clinical trials. However, the utility of these drugs has been limited by their hepatotoxicity, poor solubility, and poorly tolerated formulations. EXPERIMENTAL DESIGN: We determined the pharmacodynamic and antitumor properties of a novel, synthetic Hsp90 inhibitor, SNX-2112, in cell culture and xenograft models of HER kinase-dependent cancers. RESULTS: We show in a panel of tumor cell lines that SNX-2112 and its prodrug SNX-5542 are Hsp90 inhibitors with properties and potency similar to that of 17-AAG, including: degradation of HER2, mutant epidermal growth factor receptor, and other client proteins, inhibition of extracellular signal-regulated kinase and Akt activation, and induction of a Rb-dependent G(1) arrest with subsequent apoptosis. SNX-5542 can be administered to mice orally on a daily schedule. Following oral administration, SNX-5542 is rapidly converted to SNX-2112, which accumulates in tumors relative to normal tissues. A single dose of SNX-5542 causes HER2 degradation and inhibits its downstream signaling for up to 24 h, and daily dosing results in regression of HER2-dependent xenografts. SNX-5542 also shows greater activity than 17-AAG in a non-small cell lung cancer xenograft model expressing mutant EGFR. CONCLUSIONS: These results suggest that Hsp90 inhibition with SNX-2112 (delivered as a prodrug) may represent a promising therapeutic strategy for tumors whose growth and survival is dependent on Hsp90 clients.

The use of high-content screening for the discovery and characterization of compounds that modulate mitotic index and cell cycle progression by differing mechanisms of action.

The advent of high-content screening has expanded the ability of researchers to identify and quantify compound effects on a number of cellular events in a manner that allows for the rapid screening of chemical libraries. We have validated an approach for characterizing inhibitors of Aurora kinase family members using high-content screening by determining compound effects on the levels of the mitotic marker phospho-histone H3 (Ser10). Analysis of the data from these experiments led us to the discovery of a series of related compounds that increased the level of cells staining positive for the mitotic marker, indicating a block of cell cycle progression at M-phase. We then reconfigured the Aurora kinase inhibition assay and validated a high-content approach to identify compounds that block progression through M-phase. We were able to take advantage of the flexibility within the high-content screening platform to measure DNA content, activation of apoptosis, and effects on beta-tubulin staining patterns, all of which directly led to the identification of the cellular target of this new class of compounds.

Application of chemoproteomics to drug discovery: identification of a clinical candidate targeting hsp90.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.