title: Development of calpain-specific inactivators by screening of positional scanning epoxide libraries.
abstract: 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 mu-calpain rapidly (k(2)/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 mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-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 gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Cuerrier
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Tudor
authors.initials: T
authors.lastname: Moldoveanu
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Campbell
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Kelly
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Bilge
authors.initials: B
authors.lastname: Yoruk
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
authors.forename: Steven H L
authors.initials: SHL
authors.lastname: Verhelst
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
authors.forename: Doron
authors.initials: D
authors.lastname: Greenbaum
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada; Protein Function Discovery Group, Queen's University, Kingston, Ontario K7L 3N6, Canada. Electronic address: daviesp@post.queensu.ca.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D018360:Crystallography, X-Ray; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis*; D004852:Epoxy Compounds / Q000494:pharmacology*; D006023:Glycoproteins / Q000138:chemical synthesis*; D018716:Molecular Mimicry; D051381:Rats
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004852:Epoxy Compounds; D006023:Glycoproteins; C039304:calpain inhibitors; D002403:Cathepsins; D002154:Calpain