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Jeanne A. Hardy,
Jack T. Nguyen,
Tom O'Brien, and
James A. Wells
Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, CA 94080
Communicated by Robert M. Stroud, University of California, San Francisco, CA, July 2, 2004
Received for publication May 11, 2004.
Allosteric regulation of proteins by conformational change isa primary means of biological control. Traditionally it hasbeen difficult to identify and characterize novel allostericsites and ligands that freeze these conformational states. Wepresent a site-directed approach using Tethering for trappinginhibitory small molecules at sites away from the active siteby reversible disulfide bond formation. We screened a libraryof 10,000 thiol-containing compounds against accessible cysteinesof two members of the caspase family of proteases, caspase-3and -7. We discovered a previously unreported and conservedallosteric site in a deep cavity at the dimer interface 14 Åfrom the active site. This site contains a natural cysteinethat, when disulfide-bonded with either of two specific compounds,inactivates these proteases. The allosteric site is functionallycoupled to the active site, such that binding of the compoundsat the allosteric site prevents peptide binding at the activesite. The x-ray crystal structures of caspase-7 bound by eithercompound demonstrates that they inhibit caspase-7 by trappinga zymogen-like conformation. This approach may be useful toidentify new allosteric sites from natural or engineered cysteines,to study allosteric transitions in proteins, and to nucleatedrug discovery efforts.
Freely available online through the PNAS open access option.