Mechanistic Insights into SARS-CoV-2 Main Protease Inhibition Reveals Hotspot Residues

The main protease (M^pro) is a key enzyme responsible for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication that causes the spread of the global pandemic novel coronavirus (nCOVID-19) infection. In the present study, multiple computational approaches such as docking, long-range molecular dynamics (MD) simulations, and binding free-energy (BFE) estimation techniques were employed to investigate the mechanistic basis of the high-affinity inhibitors─GC-376, Calpain XII, and Calpain II (hereafter Calpain as Cal) from the literature─binding to M^pro. Redocking GC-376 and docking Cal XII and Cal II inhibitors to M^pro were able to reproduce all crucial interactions like the X-ray conformation. Subsequently, the apo (ligand-free) and three holo (ligand-bound) complexes were subjected to extensive MD simulations, which revealed that the ligand binding did not alter the overall M^pro structural features, whereas the heatmap analysis showed that the residues located in subsites S1 and S2, the catalytic dyad, and the ⁴⁵TSEDMLN⁵¹ loop in M^pro exhibit a conformational deviation. Moreover, the BFE estimation method was used to elucidate the crucial thermodynamic properties, which revealed that Coulomb, solvation surface accessibility (Solv_SA), and lipophilic components contributed significant energies for complex formation. The decomposition of the total BFE to per-residue showed that H41, H163, M165, Q166, and Q189 residues contributed maximum energies. The overall results from the current investigation might be valuable for designing novel anti-M^pro inhibitors.