Abstract: H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS-CoV-2 main protease through a nonnative hydrogen bond Vinicius de Oliveira University of Maryland School of Pharmacy Mohamed Ibrahim University of Luebeck Xinyuanyuan Sun University of Luebeck Rolf Hilgenfeld University of Luebeck Jana Shen (  jana.shen@rx.umaryland.edu ) University of Maryland School of Pharmacy https://orcid.org/0000-0002-3234-0769 Article Keywords: Posted Date: August 9th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1915291/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS-CoV-2 main protease through a nonnative hydrogen bond Vinicius Martins de Oliveira,† Mohamed Fourad Ibrahim,‡ Xinyuanyuan Sun,‡ Rolf Hilgenfeld,‡,¶ and Jana Shen∗,† † Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland, USA ‡ Institute of Molecular Medicine, University of Lübeck, Lübeck, Germany ¶ German Center for Infection Research (DZIF), Hamburg – Lübeck – Borstel – Riems Site, University of Lübeck, Lübeck, Germany E-mail: jana.shen@rx.umaryland.edu 1 Abstract Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir’s inhibitory activity. As the selective pressure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of the H172Y mutation on Mpro’s structure, function, and drug resistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nirmatrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H-bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir’s lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turn explains the drastic reduction in catalytic activity and antiviral susceptibility.