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

The main protease (M ^pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of M ^pro , a cysteine protease, have been determined, facilitating structure-based drug design. M ^pro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, M ^pro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV M ^pro , but have yet to be investigated for SARS-CoV-2. In this work we have 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. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M ^pro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N _δ (HD) and N _ϵ (HE) protonation of His41 and His164, respectively, the α -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately 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.