Computational and Experimental Study of the Conformational Variation of the Catalytic Residue His41 of the SARS-CoV-2 Main Protease

The main protease (M^pro) is essential for the replication of SARS-CoV-2, making it one of the major therapeutic targets for COVID-19 treatment. Here, we explored the conformational dynamics and energetics of the catalytic residue His41 in M^pro, as revealed by a rare conformational shift observed in the cocrystal structures of M^pro bound by certain inhibitors. Using steered molecular dynamics combined with umbrella sampling, we demonstrated that π-cation interactions between these inhibitors and the ionized catalytic dyad significantly reduced the energy barrier for the conformational flip of the His41 side chain. To further investigate the structure-activity relationship linked to this conformational change, we designed and synthesized a series of covalent inhibitors that control His41 flipping. Among these, compound H102-7 exhibited remarkable inhibitory activity with an IC₅₀ of 5 nM. Drug resistance studies revealed that these inhibitors displayed improved resistance profiles compared to the clinically approved M^pro covalent inhibitor, Nirmatrelvir. This study integrates computational simulations, medicinal chemistry, and molecular biology to uncover an interesting allosteric effect of a key catalytic residue of SARS-CoV-2 M^pro and yields new promising molecules for the further development of M^pro-targeted therapeutic intervention.