Exploring multiple reaction pathways in the proteolysis of SARS-CoV-2 main protease through large-scale quantum molecular dynamics

Elucidation of the enzymatic reaction mechanism of the main protease (Mpro) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is significant for the development of peptidomimetic covalent inhibitors against coronavirus disease 2019 (COVID-19) such as nirmatrelvir. Large-scale quantum molecular dynamics and metadynamics simulations were performed for wild-type Mpro dimer, monomer, and nirmatrelvir-resistant mutant with natural substrate models. The study primarily focused on the acylation stage of the catalytic cycle, which involves covalent bond formation between the enzyme and the ligand. Free energy analyses revealed that nucleophilic acyl substitution, characterized by intra-protein proton transfer, nucleophilic substitution, and proton transfer between the protein and substrate, occurs via two pathways with comparable free energy barriers in the wild-type Mpro dimer. The study revealed that the free energy barriers for the two reaction pathways in the monomer were marginally higher than those in the dimer, suggesting that dimerization contributes to the stabilization of the active-site structure, which is crucial for facilitating chemical reactions. Additionally, the nirmatrelvir-resistant mutant was observed to destabilize the intermediate state during the nucleophilic substitution process. This finding illustrates that despite the mutation conferring drug resistance, the enzymatic reactivity is preserved by altering the reaction mechanism, thereby maintaining the protease’s functionality.