In situ assembly of enzyme inhibitors using extended tethering


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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Figure 1
Figure 2: Extended tethering identifies small chemical fragments.
Figure 3: X-ray crystal structures of caspase-3 in complex with various ligands.
Figure 4: Evolution of selected fragments into reversible inhibitors of caspase-3.
Figure 5: Compound 7 inhibits Fas-induced apoptosis in Jurkat cells.

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This research was supported in part by SBIR grant no. 2 R44 CA 85141-03. We thank the staff at SSRL beamline 7.1 for assistance with data collection; Andrew C. Braisted, Darren R. Raphael, and Jeff Jacobs for monophore synthesis; the automation group (Stuart Lam, Thomas Webb, and Alex Hsi) for their help in preparatory HPLC purifications; Simone Evarts and Mark Cancilla for HRMS determinations; James A. Wells for helpful discussions; Jennifer Wilkinson for tissue culture assistance; and Monya L. Baker, Willard Lew, and Robert S. McDowell for critical readings of the manuscript.

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Correspondence to Daniel A. Erlanson or Tom O'Brien.

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Erlanson, D., Lam, J., Wiesmann, C. et al. In situ assembly of enzyme inhibitors using extended tethering. Nat Biotechnol 21, 308–314 (2003).

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