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

journal contribution
posted on 2023-06-08, 14:43 authored by Martin G Rowlands, Yvette M Newbatt, Chrisostomos ProdromouChrisostomos Prodromou, Laurence PearlLaurence Pearl, Paul Workman, Wynne Aherne
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.

History

Publication status

  • Published

Journal

Analytical Biochemistry

ISSN

0003-2697

Publisher

Elsevier Masson

Issue

2

Volume

327

Page range

176-183

Department affiliated with

  • Biochemistry Publications

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2015-02-25