title: A time-resolved fluorescence resonance energy transfer-based HTS assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors.
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.
authors.affiliation: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
authors.forename: Vicki
authors.initials: V
authors.lastname: Zhou
authors.forename: Shulin
authors.initials: S
authors.lastname: Han
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker
authors.forename: Heath
authors.initials: H
authors.lastname: Klock
authors.forename: Jeremy
authors.initials: J
authors.lastname: Caldwell
authors.forename: Xiang-ju
authors.initials: XJ
authors.lastname: Gu
mesh_terms: D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; D001710:Biotin