Nature Biotechnology21, 308 - 314 (2003)
Published online: 3 February 2003; | doi:10.1038/nbt786
In situ assembly of enzyme inhibitors using extended tethering
Daniel A. Erlanson, Joni W. Lam, Christian Wiesmann, Tinh N. Luong, Robert L. Simmons, Warren L. DeLano, Ingrid C. Choong, Matthew T. Burdett, W. Michael Flanagan, Dennis Lee, Eric M. Gordon
& Tom O'Brien
Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, CA 94080.
Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.
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