Title | A fluorescence polarization assay for inhibitors of Hsp90 |
Publication Type | Journal Article |
Year of Publication | 2006 |
Authors | Howes, R, Barril X, Dymock BW, Grant K, Northfield CJ, Robertson AG, Surgenor A, Wayne J, Wright L, James K, Matthews T, Cheung KM, McDonald E, Workman P, Drysdale MJ |
Journal | Analytical Biochemistry |
Volume | 350 |
Issue | 2 |
Pagination | 202 - 213 |
Date Published | 2006/03/15/ |
Keywords | Adenosine Triphosphatases/antagonists & inhibitors; Binding Sites; Coumarins/chemistry/pharmacology; Crystallography, X-Ray; Fluorescence Polarization/methods; HSP90 Heat-Shock Proteins/antagonists & inhibitors; Inhibitory Concentration 50; Pyrazoles/chemistry/pharmacology; Resorcinols/chemistry; Saccharomyces cerevisiae/enzymology; Structure-Activity Relationship |
Abstract | Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors. |