A fluorescence polarization assay for inhibitors of Hsp90.
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.
Howes
R
R
Vernalis (Cambridge), Granta Park, Great Abington, Cambridge CB1 6GB, UK. r.howes@vernalis.com
Barril
X
X
Dymock
B W
BW
Grant
K
K
Northfield
C J
CJ
Robertson
A G S
AG
Surgenor
A
A
Wayne
J
J
Wright
L
L
James
K
K
Matthews
T
T
Cheung
K-M
KM
McDonald
E
E
Workman
P
P
Drysdale
M J
MJ
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
D003374:Coumarins; D018841:HSP90 Heat-Shock Proteins; D011720:Pyrazoles; D012118:Resorcinols; C510240:VER-00045864; D000251:Adenosine Triphosphatases