title: A fluorescence polarization assay for inhibitors of Hsp90.
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.
authors.affiliation: Vernalis (Cambridge), Granta Park, Great Abington, Cambridge CB1 6GB, UK. r.howes@vernalis.com
authors.forename: R
authors.initials: R
authors.lastname: Howes
authors.forename: X
authors.initials: X
authors.lastname: Barril
authors.forename: B W
authors.initials: BW
authors.lastname: Dymock
authors.forename: K
authors.initials: K
authors.lastname: Grant
authors.forename: C J
authors.initials: CJ
authors.lastname: Northfield
authors.forename: A G S
authors.initials: AG
authors.lastname: Robertson
authors.forename: A
authors.initials: A
authors.lastname: Surgenor
authors.forename: J
authors.initials: J
authors.lastname: Wayne
authors.forename: L
authors.initials: L
authors.lastname: Wright
authors.forename: K
authors.initials: K
authors.lastname: James
authors.forename: T
authors.initials: T
authors.lastname: Matthews
authors.forename: K-M
authors.initials: KM
authors.lastname: Cheung
authors.forename: E
authors.initials: E
authors.lastname: McDonald
authors.forename: P
authors.initials: P
authors.lastname: Workman
authors.forename: M J
authors.initials: MJ
authors.lastname: Drysdale
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization / Q000379:methods*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D020128:Inhibitory Concentration 50; D011720:Pyrazoles / Q000737:chemistry* / Q000494:pharmacology*; D012118:Resorcinols / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D003374:Coumarins; D018841:HSP90 Heat-Shock Proteins; D011720:Pyrazoles; D012118:Resorcinols; C510240:VER-00045864; D000251:Adenosine Triphosphatases