An AlphaScreen TM -Based High-Throughput Screen to Identify Inhibitors of Hsp90-Cochaperone Interaction

Abstract

Hsp90 has emerged as an important anticancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. The authors describe the development of the first high-throughput screen, based on AlphaScreen™ technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay used the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC₅₀s in good agreement with reported values. The assay was stable over 12 h and tolerated DMSO up to 5%. The authors first validated the assay by screening against 20,000 compounds in a 384-well format. After further optimization into a 1536-well format, it was screened against an NIH Chemical Genomics Center library of 76,134 compounds, with a signal-to-background ratio of 78 and Z′ factor of 0.77. The present assay can be used for discovery of novel small-molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anticancer drugs, for use alone or in combination with other Hsp90 inhibitors. (Journal of Biomolecular Screening 2009:273-281)

Keywords

heat shock protein 90 (Hsp90)Hsp organizing protein (HOP)tetratricopeptide repeat (TPR)AlphaScreen™high-throughput screening (HTS)

References

Neckers L. : Heat shock protein 90: the cancer chaperone. J Biosci 2007;32:517-530.

Powers MP , Workman P. : Inhibitors of the heat shock response: biology and pharmacology. FEBS Lett 2007;581:3758-3769.

Chiosis G. , Timaul MN , Lucas B. , Munster PN , Zheng, FF , Sepp-Lorenzino L. , et al: A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells. Chem Biol 2001;8:289-299.

Hardcastle A. , Tomlin P. , Norris C. , Richards J. , Cordwell M. , Boxall K. , et al: A duplexed phenotypic screen for the simultaneous detection of inhibitors of the molecular chaperone heat shock protein 90 and modulators of cellular acetylation. Mol Cancer Ther 2001;6:1112-1122.

Goetz MP , Toft D. , Reid J. , Ames M. , Stensgard B. , Safgren S. , et al: Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer. J Clin Oncol 2005;23:1076-1087.

Solit DB , Ivy SP , Kopil C. , Sikorski R. , Morris MJ , Slovin SF , et al: Phase I trial of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer. Clin Cancer Res 2007 ;13:1775-1782.

Ramanathan RK , Egorin MJ , Eiseman JL , Ramalingam S. , Friedland D. , Agarwala SS , et al: Phase I and pharmacodynamic study of 17-(allylamino)-17-demethoxygeldanamycin in adult patients with refractory advanced cancers. Clin Cancer Res 2007;13:1769-1774.

Caplan AJ : What is a cochaperone? Cell Stress Chaperones 2003;8: 105-107.

Pearl LH , Prodromou C. , Workman P. : The Hsp90 molecular chaperone: an open and shut case for treatment. Biochem J 2008;3:439-453.

10.

Chen S. , Smith DF : Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery. J Biol Chem 1998;273:35194-35200.

11.

Scheufler C. , Brinker A. , Bourenkov G. , Bartunik H. , Hartl FU , Moarefi I. : Structure of TPR domain-peptide complexes: critical elements of the assembly of the Hsp70-Hsp90 multichaperone machine . Cell 2000;101: 199-210.

12.

Brinker A. , Scheufler C. , von der Mulbe F. , Fleckenstein B. , Herrmann C. , Jung G. , et al: Ligand discrimination by TPR domains: relevance and selectivity of EEVD-recognition in Hsp90-HOP-Hsp90 complexes. J Biol Chem 2002;277:19265-19275.

13.

Chen MS , Silverstein AM , Pratt WB , Chinkers M. : The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant. J Biol Chem 1996;271:32315-32320.

14.

Cortajarena AL , Yi F. , Regan L. : Designed TPR modules as novel anticancer agents. ACS Chem Biol 2008;3:161-166.

15.

Ullman EF , Kirakossian H. , Singh S. , Wu ZP , Irvin BR , Pease PS , et al: Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci USA 1994;91: 5426-5430.

16.

Warner G. , Illy C. , Pedro L. , Roby P. , Bosse R. : AlphaScreen kinase HTS platforms. Curr Med Chem 2004;11:721-730.

17.

Inglese J. , Auld DS , Jadhav A. , Johnson RL , Simeonov A. , Yasgar A. , et al: Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries . Proc Natl Acad Sci USA 2006;103:11473-11478.

18.

Feng BY , Shoichet BK : A detergent-based assay for the detection of promiscuous inhibitors. Nat Protocols 2002;1:550-553.

19.

McGovern SL , Helfand BT , Feng B. , Shoichet BK : A specific mechanism of nonspecific inhibition . J Med Chem 2003;46:4265-4272.

20.

Yi F. , Regan L. : A novel class of Hsp90 inhibitors. ACS Chem Biol. In press.

21.

Roe SM , Ali MM , Meyer P. , Vaughan CK , Panaretou B. , Piper PW , et al: The mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50cdc37. Cell 2004;116:87-98.

22.

Riggs DL , Cox MB , Cheung-Flynn J. , Prapapanich V. , Carrigan PE , Smith DF : Functional specificity of cochaperone interactions with Hsp90 client proteins . Crit Rev Biochem Mol Biol 2004;39:279-295.