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PNAS 101 (34): 12461-12466

Copyright © 2004 by the National Academy of Sciences.


Discovery of an allosteric site in the caspases

Jeanne A. Hardy, Joni Lam, Jack T. Nguyen {dagger}, Tom O'Brien, and James A. Wells {ddagger}

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.

Abstract: Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 Å from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.

Freely available online through the PNAS open access option.

Abbreviations: DICA, 2-(2,4-dichloro-phenoxy)-N-(2-mercapto-ethyl)-acetamide; FICA, 5-fluoro-1H-indole-2-carboxylic acid (2-mercapto-ethyl)-amide; DEVD, Asp-Glu-Val-Asp.

Data deposition: The atomic coordinates of the DICA and FICA complexes with caspase-7 have been deposited in the Protein Data Bank, (PDB ID codes 1SHJ and 1SHL, respectively).

{dagger} Present address: Catalyst Biosciences, Inc., 225 Gateway Boulevard, South San Francisco, CA 94080.

The term Tethering is a service mark of Sunesis Pharmaceuticals for its fragment-based drug discovery method.

{ddagger} To whom correspondence should be addressed. E-mail: jaw{at}

© 2004 by The National Academy of Sciences of the USA

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