The main protease (M^pro) 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 M^pro. The M^pro 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 M^pro, 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 M^pro 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 M^proare 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 M^pro 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.

The arrangement of histidines in the active site of SARS-CoV-2 M<sup>pro</sup>.

The arrangement of histidines in the active site of SARS-CoV-2 M^pro.

Anna Pavlova

Georgia Institute of Technology

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