Comparative docking and molecular dynamics studies of molnupiravir (EIDD-2801): implications for novel mechanisms of action on influenza and SARS-CoV-2 protein targets

AbstractMolnupiravir (EIDD-2801) (MLN) is an oral antiviral drug for COVID-19 treatment, being integrated into viral RNA through RNA-dependent RNA polymerase (RdRp). Upon ingestion, MLN is transformed into two active metabolites: β-d-N4-hydroxycytidine (NHC) (EIDD-1931) in the host plasma, and EIDD-1931-triphosphate (MTP) within the host cells. However, recent studies provide increasing evidence of MLN's interactions with off-target proteins beyond the viral genome, suggesting that the complete mechanisms of action of MLN remain unclear. The aim of this study was therefore to investigate the molecular interactions of MLN in the form of NHC and MTP with the non-RNA structural components of avian influenza (hemagglutinin, neuraminidase) and SARS-CoV-2 (spike glycoprotein, Mpro, and RdRp) viruses and to elucidate whether these two metabolites possess the ability to form stable complexes with these major viral components. Molecular docking of NHC and MTP was performed using AutoDock 4.2.6 and the obtained protein-drug complexes were submitted to 200-ns molecular dynamics simulations in triplicate with subsequent free energy calculations using GROMACS. Docking scores, molecular dynamics and MM/GBSA results showed that MTP was tightly bound within the active site of SARS-CoV-2 RdRp and remained highly stable throughout the 200-ns simulations. Besides, it was also shown that NHC and MTP formed moderately-to-highly stable molecular complexes with off-target receptors hemagglutinin, neuraminidase and Mpro, but rather weak interactions with spike glycoprotein. Our computational findings suggest that NHC and MTP may directly inhibit these receptors, and propose that additional studies on the off-target effects of MLN, i.e. real-time protein binding assays, should be performed.Communicated by Ramaswamy H. SarmaKeywords: MolnupiravirinfluenzaSARS-CoV-2off-target effectmolecular dockingmolecular dynamicsMM/GBSA Disclosure statementE.S.I.: conceptualized the study, performed molecular docking simulations of NHC and MTP, prepared Figures 2, 3, and Tables 1-2, as well as contributed to the writing of the manuscript. N.O.: helped in critical reviewing of the work, and provided motivation to investigate the effects of MLN on structural and non-structural proteins of SARS-CoV-2. C.S.: prepared Figure 1, and provided valuable input for the 'Discussion' section of the manuscript. P.A.N., and E.R.K.: performed 200-ns molecular dynamics simulations of NHC and MTP in triplicate, conducted MM/GBSA calculations, prepared Table 3, Figures 4, 5, and significantly aided in the writing of the manuscript. A.S.T.: Contributed significant insights for the 'Discussion' section, and aided in refining the literature.Additional informationFundingP. A. N. and E. R. K. acknowledges financial support from CNPq and CAPES [Financial code 001], as well as the Centro Nacional de Supercomputação (CESUP), Universidade Federal do Rio Grande do Sul (UFRGS) for providing HPC resources.