Efforts to identify new hsp90 inhibitors have focused on modifying three natural chemical scaffolds; geldanamycin, purine, and radicicol. Although some members of these families have progressed into clinical trials, there is a need to identify new small molecule scaffolds with improved pharmaceutical properties. An unbiased chemoproteomics-based screen was initiated to discover novel anti-tumor agents. A targeted library was designed and screened using a proprietary protein affinity-displacement assay that employed a purine-based affinity resin to capture purine-binding proteins from cells or tissues of interest. This resin was designed to reversibly bind proteins through their purine co-factor site. Screening hits were those compounds that eluted one or more proteins in a concentration-dependent manner from the resin. The primary screen provided both target affinity and selectivity, facilitating hit follow-up. In parallel, this library was screened in a small panel of cancer cell proliferation assays. Nearly 750 compounds (~10% hit rate) eluted over 75 proteins across dozens of enzyme families including kinases, chaperone proteins, sulfotransferases, and reductases. Multiple chemical scaffolds were identified with varying affinity and selectivity toward hsp90. Medicinal chemistry around one hit, SC-3423, led to the discovery of SNX-2321 whose binding mode was established through x-ray analysis of a co-crystal with the N-terminal domain of hsp90. These results demonstrated that SNX-2321 bound into the ATP site, utilizing key hydrogen-bond interactions that have previously been observed for other hsp90 inhibitors. Further optimization of this series led to the identification of SNX-7081 as a potent and highly selective hsp90 inhibitor. Against a panel of 7 tumor cell lines, SNX-7081 blocked cell proliferation with a potency of 1 - 25 nM and was 7-200 fold more potent than 17-AAG in these cell lines. Consistent with its mechanism as a selective hsp90 inhibitor, SNX-7081 induced client protein effects at concentrations comparable to those required to inhibit cell proliferation. SNX-7081 was also efficacious in vivo, providing significant inhibition of tumor growth in an HT-29 xenograft model. In conclusion, a novel chemoproteomics screen was employed to successfully generate multiple unique small molecule inhibitors of hsp90; one series was further optimized to produce highly selective inhibitors that exceed the potency of current clinical candidates.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA