A Molecular Dynamics Study of Michaelis Complex for Designing Selective Transition State Analog Inhibitors for Cysteine Protease calpain-2

Abstract

Calpains constitute a family of intracellular cysteine proteases, which catalyze the cleavage of target proteins in response to Ca2+ signaling. We recently discovered that, calpain-1 induces long-term potentiation, while calpain-2 limits the extent of potentiation and also is involved in neurodegeneration by NMDA activation. There is therefore a strong rationale to develop calpain-2 selective inhibitors, as such compounds could enhance learning and memory and offer neuroprotection. Among several reversible inhibitors that have been developed to target calpains, peptidyl α-ketoamide analogs carry the most stable electrophilic warhead. Although several cocrystal structures of calpain-1 and α-ketoamide are available, it is not clear how to design calpain-2 specific inhibitors based on the calpain-1 model, as the protease core is highly conserved. Structural alignment reveals that the major difference between calpain-1 and calpain-2 is located at the flexible gating loop. Molecular dynamics simulation of ligand-protease Michaelis complex is therefore necessary to capture the difference in the noncovalent binding interaction between the inhibitor and two isoforms and thus to guide the structure-based design. The Michaelis complex model of calpain-2 with a ketoamide compound was built from a cocrystal structure of a potent calpain-1 inhibitor. Effects of catalytic triad protonation states on the stability of the complex were investigated. The model was validated using existing calpain-2 inhibitors. Based on the simulation results, we modified the prime side (C-terminal) of the α-ketoamide peptidomimetics to favor calpain-2 over calpain-1. The dynamic of the calpain gating loop was explored using long-time scale simulation. The results show that molecular dynamics simulation of Michaelis complex can be a critical tool to explore the molecular bases of calpain activation and to design calpain-2 selective inhibitors for clinical therapeutics.