Computational Insights into the Regioselective Hydroxylation of Nirmatrelvir Metabolized by Cytochrome P450 3A4

Nirmatrelvir, a covalent inhibitor of SARS-CoV-2 main protease (M^pro), is the active pharmaceutical ingredient in Paxlovid─the first oral antiviral drug granted emergency use authorization by the U.S. Food and Drug Administration (FDA) for the treatment of COVID-19. Previous studies revealed that the hepatic metabolism of nirmatrelvir was predominantly mediated by cytochrome P450 3A4 (CYP3A4) and that nirmatrelvir can be metabolized to generate multiple products. However, the precise molecular mechanism underlying the regioselective metabolism of nirmatrelvir by CYP3A4 remains to be disclosed. In this study, we performed an integrated computational strategy combining molecular docking, molecular dynamics (MD) simulations, and quantum chemical (QC) calculations to investigate the mechanisms of interaction between CYP3A4 and nirmatrelvir. Our simulation results revealed key insights into the spatial proximity between nirmatrelvir's carbon atoms and the reactive iron(IV)-oxo species (Compound I, Cpd I), with the C23 position showing the greatest accessibility, suggesting a strong preference for this site. Density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations further demonstrated that the transition state for C23 hydroxylation possesses the lowest activation energy barrier, consistent with the experimentally observed regioselectivity in metabolite formation.