Cocrystal structures of primed side-extending α-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca2+ signaling. When Ca2+ homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases Such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (mu I-II) bound to two alpha-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward mu I-II and heterodimeric m-calpain. Moreover, stacking orients the adenine Such that it can be used its a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.