Molecular determinants of K36 binding in SARS-CoV-2 main protease revealed by MD simulations and alanine mutagenesis

The SARS-CoV-2 main protease (Mpro) is a crucial target for antiviral drugs because of its vital function in viral polyprotein processing and replication. This study utilized molecular dynamics (MD) simulations to examine the binding stability and dynamic behavior of the inhibitor K36 with the wild-type (WT) Mpro and fifteen alanine-substituted versions that target functionally significant residues. The binding affinity was assessed by MM/PBSA calculations, indicating robust binding for the WT complex with an average binding free energy of around -20.0 kJ/mol. Numerous mutations significantly diminished ligand binding, resulting in binding free energies declining to roughly -6.1 kJ/mol for the most destabilizing variations, signifying mutation-dependent disruption of ligand stability in the active site. Structural stability and conformational dynamics were assessed by RMSD, RMSF, principal component analysis, hydrogen-bond lifetime analysis, and secondary structure profiling. The WT complex exhibited steady backbone dynamics, enduring intermolecular hydrogen bonding with an average hydrogen-bond lifetime of 16.8 ps, and preserved secondary structure content across the 500 ns simulations. Conversely, mutations at critical catalytic and substrate-binding residues, such as Cys145, His163, Glu166, and His41, demonstrated diminished hydrogen bond stability and heightened conformational flexibility, aligning with impaired ligand binding. These results elucidate the mechanisms by which mutations alter Mpro ligand interactions and underscore the promise of K36 as a viable scaffold for the development of antiviral inhibitors.