The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the viral life cycle by cleaving the two SARS-CoV-2 polyproteins, making it an attractive target for antiviral therapeutics. Several promising lead compounds have already been discovered, and the release of many apo and inhibitor-bound structures have facilitated the structure-based drug design efforts targeting Mpro . The Mpro binding site contains multiple histidines, including His41 that is a part of the His41-Cys145 catalytic dyad. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in earlier studies of SARS-CoV Mpro, but they have not been investigated for SARS-CoV-2. Here, we used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues in both the apo and inhibitor-bound enzyme. It is found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these protonation assignments. Surprisingly, distinct protonation state stabilities were observed for the two studied inhibitors: the peptidomimetic N3 and a ketoamide. Our results demonstrate the importance of using accurate histidine protonation states to model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts. We also performed longer simulations of the inhibitor-bound and apo structures, using structurally optimal protonation states, and the stability of different inhibitor classes was investigated.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease

ACS Spring Meeting 2021

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