Abstract 1789: Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform

Abstract HSP90 is a ubiquitous molecular chaperone that couples ATP hydrolysis to the folding and maturation of numerous client proteins. Because many of these client proteins are essential to cancer cell growth and survival, HSP90 inhibitors have undergone clinical investigation in numerous oncology indications. Here, using an LC-MS/MS platform previously developed to characterize ATP-competitive inhibitors of protein kinases, several HSP90 inhibitors were profiled in native cell lysates. This method utilizes an ATP derivative that covalently labels lysine residues in the ATP-binding site of any amenable enzyme. The specificity of inhibitors for all four paralogs of HSP90 (HSP90α, HSP90β, Grp94 and Trap-1) found in human cells was simultaneously monitored at their endogenous relative abundances. Interestingly, the concentration of HSP90 paralogs in human cell lysates was significantly higher than reported inhibitor binding constants, therefore complete inhibition was only observed at much higher concentrations than would have been predicted. In addition to the four HSP90 paralogs, inhibition data was collected on over 400 protein kinases and other nucleotide-binding proteins. This led to the discovery that NVP-AUY922 has off-target activity toward PMS2, an enzyme, like HSP90 itself, belonging to the GHKL ATPase superfamily. In addition to directly profiling compound targets in lysates, downstream effects of HSP90 inhibition were monitored by treating live cells and analyzing the resulting cell lysates at defined time points. Many proteins were found to be much less abundant in the compound-treated cells, including known HSP90 client proteins and novel, putative client proteins. Citation Format: Tyzoon K. Nomanbhoy, Brian E. Nordin, Jonathan Rosenblum, Yongsheng Liu. Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2014-1789