title: High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.
abstract: The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Rowlands
authors.forename: Yvette M
authors.initials: YM
authors.lastname: Newbatt
authors.forename: Chrisostomos
authors.initials: C
authors.lastname: Prodromou
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
authors.forename: Wynne
authors.initials: W
authors.lastname: Aherne
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003124:Colorimetry; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000032:analysis
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D018841:HSP90 Heat-Shock Proteins; D010710:Phosphates; D000251:Adenosine Triphosphatases