Rational Design of Calpain Inhibitors Based on Calpastatin Peptidomimetics

Our
previously reported structures of calpain bound to its endogenous
inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated
peptide macrocyclization toward the design of cyclic peptides and
peptidomimetics as calpain inhibitors. Inspired by nature’s
hint that a β-turn loop within calpastatin forms a broad interaction
around calpain’s active site cysteine, we have constructed
and tested a library of 45 peptidic compounds based on this loop sequence.
Four molecules have shown reproducibly low micromolar inhibition of
calpain-2. Further systematic sequence changes led to the development
of probes that displayed increased potency and specificity of inhibition
against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling
other peptidomimetic calpain inhibitors and presenting an improved
selectivity profile against other therapeutically relevant proteases.
Competitive and mixed inhibition against calpain-2 was observed, and
an allosteric inhibition site on the enzyme was identified for a noncompetitive
inhibitor.