Slow, tight binding inhibition of Hsp90 isoforms correlates with potent anti-proliferative activity in cancer cell lines

3943 As the molecular chaperone Hsp90 has emerged as a promising therapeutic target for the treatment of cancer, attention has increasingly focused on the biochemical mechanism of its inhibition. Most of this attention has focused on the apparent inconsistency between the low micromolar in vitro Hsp90 binding potency of geldanamycin and its analogs compared to their low nanomolar activity against cultured cancer cells. Many hypotheses have been put forward to explain this inconsistency including differences between purified and complexed Hsp90, differences in affinity between normal and tumor Hsp90, reduction of the analogs to their dihydroquinones, differential activity against the 4 Hsp90 family members and unique kinetics of Hsp90 inhibition. We present here further evidence of a time dependent shift in affinity for several ATP competitive Hsp90 inhibitors. This time-dependent shift in affinity correlates with the anti-proliferative activity of these compounds against cancer cell lines. In particular, we describe the development of a proteomics-based assay that profiles the ability of compounds to competitively bind to the ATP pocket of all Hsp90 isoforms in a time-dependent manner, the first reported assay for all Hsp90’s. This assay was applied to geldanamycin analogs, radicicol analogs, purine analogs and several of Serenex’s novel Hsp90 inhibitors. While geldanamycins and the Serenex inhibitors exhibit profound time-dependent binding, other classes of Hsp90 inhibitors do not. In fact, geldanamycin and SNX-2112 show an 11-fold and 14-fold increase in potency respectively when drug incubation time is increased from 5 minutes to 1.5 hours. In addition, analysis of the extensive SAR set of Serenex small molecule Hsp90 inhibitors as well as geldanamycin and its analogs reveal that the kinetics of binding can discriminate between Hsp90 family members and thereby create selectivity within the Hsp90 axis. This assay has enabled the identification of compounds with optimal selectivity and binding kinetics and demonstrates that these kinetics correlate with increased anti-proliferative potency against cancer cell lines. Indeed, unlike geldanamycin and its analogs, the Serenex clinical molecule, SNX-2112, is highly potent against the alpha and beta isoforms of Hsp90 but lacks significant activity against the others, Grp94 and Trap1. A model for the modulation of binding kinetics based on x-ray co-crystal data is presented.