Document Detail

High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.
MedLine Citation:
PMID:  15051534     Owner:  NLM     Status:  MEDLINE    
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.
Martin G Rowlands; Yvette M Newbatt; Chrisostomos Prodromou; Laurence H Pearl; Paul Workman; Wynne Aherne
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  327     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2004 Apr 
Date Detail:
Created Date:  2004-03-30     Completed Date:  2004-11-29     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  176-83     Citation Subset:  IM    
Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK.
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MeSH Terms
Adenosine Triphosphatases / antagonists & inhibitors*,  metabolism
Drug Screening Assays, Antitumor / methods*
Enzyme Inhibitors / pharmacology
Fungal Proteins / isolation & purification,  metabolism
HSP90 Heat-Shock Proteins / antagonists & inhibitors*,  isolation & purification,  metabolism
Phosphates / analysis
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Fungal Proteins; 0/HSP90 Heat-Shock Proteins; 0/Phosphates; EC 3.6.1.-/Adenosine Triphosphatases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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