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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
Full text not available from this repository.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 |