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J. Biol. Chem., Vol. 282, Issue 13, 9600-9611, March 30, 2007

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Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries^*

Dominic Cuerrier¹, Tudor Moldoveanu², Robert L. Campbell, Jacqueline Kelly, Bilge Yoruk, Steven H. L. Verhelst, Doron Greenbaum³, Matthew Bogyo, and Peter L. Davies^¶4

From the Department of Biochemistry and ^¶Protein Function Discovery Group, Queen's University, Kingston, Ontario K7L 3N6, Canada and the Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and µ-calpain rapidly (k₂/K_i = 131,000 and 16,500 M^–1 s^–1, respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat µ-calpain inactivated by the epoxide compounds WR -cyano--aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR -cyano--aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

Received for publication, November 7, 2006 , and in revised form, December 12, 2006.

^* This work was supported in part by grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research (to P. L. D.), National Technology Center for Networks and Pathways Grant U54 RR020843 from the National Institutes of Health, and National Institutes of Health Grant R01-EB005011 (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2NQG and 2NQI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Supported by the Government of Canada Natural Sciences and Engineering Research Council studentship.

² Holds a Canadian Institutes of Health Research postdoctoral fellowship. Present address: Dept. of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105.

³ Present address: Dept. of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6160.

⁴ Holds a Canada Research Chair in Protein Engineering. To whom correspondence should be addressed: Dept. of Biochemistry, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2984; Fax: 613-533-2497; E-mail: daviesp{at}post.queensu.ca.

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