Development of calpain-specific inactivators by screening of positional scanning epoxide libraries.
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.
Cuerrier
Dominic
D
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Moldoveanu
Tudor
T
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Campbell
Robert L
RL
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Kelly
Jacqueline
J
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Yoruk
Bilge
B
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Verhelst
Steven H L
SHL
Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
Greenbaum
Doron
D
Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
Bogyo
Matthew
M
Department of Pathology, Stanford University School of Medicine, Stanford, Califorina 94305.
Davies
Peter L
PL
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.
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
D015853:Cysteine Proteinase Inhibitors; D004852:Epoxy Compounds; D006023:Glycoproteins; C039304:calpain inhibitors; D002403:Cathepsins; D002154:Calpain