title: Improvement of inhibitor identification for heat shock protein 90α by utilizing a red-shifted fluorescence polarization probe.
abstract: Heat shock protein-90 (HSP90) is an ATP-dependent molecular chaperone with intrinsic ATPase activity. HSP90 is required for the stability and function of client proteins, many of which are involved in oncogenesis. Thus, identification of HSP90 inhibitors would potentially lead to the discovery of cancer therapeutics. Here, we present a high-throughput screening campaign utilizing two geldanamycin (GM)-labeled probes in a fluorescence polarization (FP) assay. For the primary screen, a previously reported green BODIPY-labeled GM (GM-BODIPY) was used to evaluate a library collection of about 400,000 compounds. From this screen, 3058 compounds showed >30% inhibition. To distinguish true positives from compound interference, a confirmatory screen was deemed necessary. Accordingly, a red-shifted FP binding assay was developed using GM labeled with red BODIPY. This tool enabled reliable identification of promising HSP90α inhibitors.
authors.affiliation: Lead Discovery and Profiling, Worldwide Discovery Research, Cephalon, Inc., West Chester, Pennsylvania 19380, USA. jie.qian@reactionbiology.com
authors.forename: Jie
authors.initials: J
authors.lastname: Qian
authors.forename: Beverly P
authors.initials: BP
authors.lastname: Holskin
authors.forename: Jay
authors.initials: J
authors.lastname: Theroff
authors.forename: Ted
authors.initials: T
authors.lastname: Underiner
authors.forename: Sheryl L
authors.initials: SL
authors.lastname: Meyer
authors.forename: Thelma S
authors.initials: TS
authors.lastname: Angeles
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D001896:Boron Compounds / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D015870:Gene Expression; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D047029:Lactams, Macrocyclic / Q000494:pharmacology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C523490:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-geldanamycin; D016227:Benzoquinones; D001896:Boron Compounds; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D047029:Lactams, Macrocyclic