Molecular simulations have been instrumental in elucidating the SARS-CoV-2 lifecycle, thereby supporting the design and development of antiviral therapies and diagnostic tools for COVID-19. Here, the molecular mechanism of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), a potential target for antiviral drugs to treat COVID-19, was explored based on QM/MM simulations with fixed-charge and polarizable force fields (cFF and pFF, respectively). The free energy perturbation (FEP) method allowed exploring the free energy landscape of the enzymatic reaction mechanism, addressing key questions about the initial deprotonation of the 3′-OH group of the terminal nucleotide before a nucleophilic attack on the incoming nucleotide takes place. Indeed, among the five mechanisms explored, the most favorable was identified as a three-step process. The first step consists of a proton transfer from the 3′-OH group of the terminal nucleotide to a hydroxide group coordinated with an Mg2+ ion. Subsequently, the O3′ atom nucleophilically attacks the Pα atom of the incoming ATP. Finally, a proton is transferred from the water molecule formed in the first step to the γ-phosphate group of the pyrophosphate leaving group, regenerating the Mg2+-coordinated hydroxide group. This mechanism was found to be exergonic, with the rate-determining step being the nucleophilic attack, having a free energy barrier of 15.2 kcal mol–1. Both cFF and pFF yield consistent energetic and geometrical descriptions of the full RdRp-catalyzed reaction. Noncovalent interaction (NCI) and electron localization function (ELF) analyses provide insights into the electronic evolution during the reaction, showing strong polarization on electronic basins associated with the reactive oxygens O3′ and O3α. Together, findings contribute to a deeper understanding of the RdRp mechanism, which could aid in the discovery of new antiviral inhibitors.

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Molecular simulations have been instrumental in elucidating the SARS-CoV-2 lifecycle, thereby supporting the design and development of antiviral therapies and diagnostic tools for COVID-19. Here, the ...

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