Development of α-Helical Calpain Probes by Mimicking a Natural Protein–Protein Interaction

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening twenty-four commercially available crosslinkers for successful cysteine alkylation in a model peptide sequence. The effects of crosslinking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid crosslinkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs and will be useful for future investigation of calpains, while the crosslinking technique will enable exploration of other protein-protein interactions