In Silico Screening for Small Molecules to Alter Calpain Proteolysis through Modulating Conformation Changes Induced...

Dysregulated calpain-1 and calpain-2 protease activity linked to several diseases has encouraged efforts to explore inhibiting calpain to provide therapeutic benefits. However, there are currently no clinically approved drugs that specifically target calpain functionality. To address this unmet need, we carried out in silico drug discovery efforts to identify small molecules capable of modulating calpain activity. Our approach is based on the observation that heterodimer formation of the calpain-2 catalytic (CAPN2) and regulatory (CAPNS1) subunits is needed for both proteolytic activity and CAPN2 stability. In recognition of this obligate protein-protein interaction (PPI), the CAPN2-CAPNS1 interface was targeted with nearly 3.6 million small molecules to find candidates that bind at the interface with high affinity and introduce steric clashes capable of altering heterodimerization or the conformation of CAPN2, thereby modulating proteolytic activity. Twenty small molecules predicted to disrupt the most hydrogen bonds at the CAPN2-CAPNS1 interface were validated experimentally. Five small molecules inhibited calpain activity by 53.6 ± 4.1, 36.8 ± 38.3, 31.1 ± 17.5, 69.8 ± 27.3, and 47.1 ± 18.5%, while two enhanced protease activity by 163.0 ± 41.9 and 129.2 ± 11.9%. Unexpectedly, the effects of these seven molecules on the CAPN2-CAPNS1 PPI assay did not correlate with their effects on protease activity. Molecular simulation showed that small molecules that modulate calpain activity without abolishing heterodimerization do so by altering the conformational changes needed for the activity. This apparent allosteric mechanism paves the way for developing novel therapeutic solutions for modulating the calpain activity in various diseases associated with calpain dysregulation.