Zhou, Vicki, Han, Shulin, Brinker, Achim, Klock, Heath, Caldwell, Jeremy and Gu, Xiang-Ju (2004) A time-resolved fluorescence resonance energy transfer-based HTS assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors. Analytical Biochemistry, 331 (2). pp. 349-357. ISSN 0003-2697

Abstract

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone required for the stability and function of a number of client proteins, many of which are involved in cancer development. The natural products geldanamycin (GM) and radicicol (RD) are known inhibitors of Hsp90, and their derivatives are being developed for the treatment of various cancers. To identify novel Hsp90 inhibitors, a highly robust time-resolved fluorescence resonance energy transfer (TR-FRET)-based HTS assay that measures the binding of biotinylated geldanamycin (biotin-GM) to the His-tagged human Hsp90 N-terminal ATP-binding domain (Hsp90N) was developed. This assay was optimized in 1536-well plates and was used as the primary assay to screen 10(6) compounds. Identified "hits" were then confirmed in a scintillation proximity assay (SPA) and a DEAE membrane-based assay for [(3)H]AAG binding to Hsp90. In addition, a surface plasmon resonance (SPR) assay that measures the direct interaction of Hsp90 with its inhibitors was developed and used to further characterize the identified inhibitors. Several potent and reversible inhibitors of human Hsp90 with K(d) values measured in the high nanomolar range were identified.

Item Type:	Article
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2004.04.011
Additional Information:	author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords:	Hsp90; Binding assay; TR-FRET; SPA; SPR; Biotin-GM; Geldanamycin
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2004.04.011
Date Deposited:	14 Dec 2009 14:02
Last Modified:	31 Jan 2013 01:21
URI:	https://oak.novartis.com/id/eprint/271