High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity

Rowlands, Martin G, Newbatt, Yvette M, Prodromou, Chrisostomos, Pearl, Laurence, Workman, Paul and Aherne, Wynne (2004) High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Analytical Biochemistry, 327 (2). pp. 176-183. ISSN 0003-2697

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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.

Item Type: Article
Keywords: Adenosine Triphosphatases/*antagonists & inhibitors/metabolism Colorimetry Drug Screening Assays, Antitumor/*methods Enzyme Inhibitors/pharmacology Fungal Proteins/isolation & purification/metabolism HSP90 Heat-Shock Proteins/*antagonists & inhibitors/isolation & purification/metabolism Phosphates/analysis
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology Including cancer and carcinogens
Depositing User: Chrisostomos Prodromou
Date Deposited: 25 Feb 2015 09:33
Last Modified: 25 Feb 2015 09:33
URI: http://sro.sussex.ac.uk/id/eprint/44373
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