| Calpains are a family of Ca2+-dependent cysteine proteases involved in intracellular signalling. Their involvement in the pathogenesis of ischemic/reperfusion injury and neurodegeneration makes them important therapeutic targets. However, the similarity of the active site of calpains to that of other intracellular cysteine proteases such as cathepsins is reflected in the poor specificity of most calpain inhibitors, which limits their value for cell studies and as potential drug leads. A better understanding of the active-site specificity of calpain is therefore necessary for the development of inhibitors with improved specificity. Mini-calpains, composed of the isolated proteolytic core of calpains, present a simplified system to study the active site of calpains. Moreover, advances in combinatorial chemistry allow large libraries of compounds to be synthesized and provide an opportunity for a comprehensive study of protease specificity. Degenerate peptide libraries were used in combination with N-terminal sequencing to obtain a complete substrate specificity profile for mini-calpain muI-II. This observed specificity was validated by the synthesis of a fluorogenic calpain substrate with superior kinetics to previously available substrates. Subsequently, the screening of positional-scanning libraries of epoxide compounds by a novel SDS-PAGE assay led to the design of a series of potent calpain inhibitors. The best compound, WRH(R,R), is incapable of inactivating cathepsins B, L and K, thus demonstrating a high specificity for calpains. In a complementary approach, X-ray crystallography of four calpain-inhibitor complexes provided atomic-level details of the interactions at the active site, leading to insights into inhibitor specificity and the description of a novel loop displacement necessary for the binding of substrates and inhibitors to the active site of calpains. |
