Abstract B99: Targeting TPR cochaperones of the Hsp90 molecular chaperone complex for specific inhibition.
Authors: Darby John F. , Vidler Lewis , Simpson Pete J. , Sharp Swee , Prodromou Chris , Matthews Steve , Hoelder Swen , Pearl Laurence , Workman Paul 
Published: June 2012

The Hsp90 molecular chaperone complex plays an important role in tumour biology by aiding oncogenic client proteins in their stability and activity. Targeted therapeutics that disrupt the Hsp90 function have shown promise in clinical trials (eg 17-allylamino-17-demethoxygeldanamycin, 17-AAG, and the synthetic resorcinylic isoxazole NVP-AUY922). These drugs bind Hsp90's ATP/ADP-site, thereby inhibiting the ATP-coupled conformational cycle which plays a crucial part of chaperoning several oncogenic client proteins. Hsp90 functions as part of a chaperone complex that requires the involvement of a diverse series of cochaperones, including the Hsp70/Hsp90 organising protein (Hop). These proteins perform regulatory roles or act as scaffolds to ensure complex stability and correct client selection. A subset of structurally related but functionally diverse cochaperones interact with the C-terminal MEEVD sequence of Hsp90 through a tetratricopeptide repeat (TPR) domain. This work aims to discover inhibitors of this interaction to allow selective inhibition of Hsp90 functions and provide chemical probes for studying cochaperone biology.

Two biochemical binding assays were developed for screening compounds for inhibition of the Hsp90-TPR interaction. The assays used AlphaScreen and LANCE technology and provided a high-throughput platform for assessing compounds for inhibition against a panel of TPR proteins. Hit compounds were validated using biophysical techniques. Inhibitor effects on TPR stability in the absence of a competing substrate were assessed using a thermal shift assay. In addition, ligand binding sites were identified using 1H-15N HSQC chemical shift mapping onto an existing crystal structure.

These robust assays allowed high-throughput screening of an 80,000 compound library with mean Z'=0.92 and CV=2.6%, giving a hit rate of 1.3%. In addition compounds identified through in silico screening methods have also been tested using these assays. Three general inhibitor chemotypes have been discovered. Chemical shift mapping has shown that although these inhibitors are weak, it is likely that several different binding sites are being exploited. Improvements in potency are being pursued through analogue by catalogue methods guided by the NMR shift mapping data.

Specific Hsp90-TPR inhibitors will provide important tools for investigating Hsp90/cochaperone complexes and the dependency of client subsets on particular cochaperones. In addition, inhibitors of some TPR cochaperones may sensitize cells to existing Hsp90 inhibitors or perhaps act as independent inhibitors of specific Hsp90 functions. This work has discovered several weak inhibitors of this interaction in a biochemical setting and characterised their interactions with Hop TPR 2A. Further optimisation could provide useful chemical probes and potential starting points for drug discovery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B99.