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.