Potency Prediction of Covalent Inhibitors against SARS-CoV-2 3CL-like Protease and Multiple Mutants by Multiscale Simulations

3-Chymotrypsin-like protease (3CLpro) is a prominent target against pathogenic coronaviruses. Expert knowledge of the cysteine-targeted covalent reaction mechanism is crucial to predict the inhibitory potency of approved inhibitors against 3CLpros of SARS-CoV-2 variants and perform structure-based drug design against newly emerging coronaviruses. We carried out an extensive array of classical and hybrid QM/MM molecular dynamics simulations to explore covalent inhibition mechanisms of five well-characterized inhibitors toward SARS-CoV-2 3CLpro and its mutants. The calculated binding affinity and reactivity of the inhibitors are highly consistent with experimental data, and the predicted inhibitory potency of the inhibitors against 3CLpro with L167F, E166V, or T21I/E166V mutant is in full agreement with IC50s determined by the accompanying enzymatic assays. The explored mechanisms unveil the impact of residue mutagenesis on structural dynamics that communicates to change not only noncovalent binding strength but also covalent reaction free energy. Such a change is inhibitor dependent, corresponding to varied levels of drug resistance of these 3CLpro mutants against nirmatrelvir and simnotrelvir and no resistance to the 11a compound. These results together suggest that the present simulations with a suitable protocol can efficiently evaluate the reactivity and potency of covalent inhibitors along with the elucidated molecular mechanisms of covalent inhibition.