1’-cyano substitution of Remdesivir “template-dependent” inhibiting the transcription of SARS-CoV-2

Remdesivir is one nucleotide analog prodrug capable to terminate RNA synthesis in SARS-CoV-2 RdRp by two distinct mechanisms. The “delayed chain termination mechanism” has been extensively investigated, while the “template-dependent inhibition mechanism” remains elusive. In this study, we have demonstrated that Remdesivir embedded in the template strand seldom directly disrupted the complementary NTP incorporation at the active site. Instead, the translocation of the template strand from +2 to +1 site was hindered, as the 1’-cyano group of Remdesivir would sterically clash with V557. Moreover, we have elucidated the molecular mechanism of how SARS-CoV-2 RdRp gained the drug resistance to Remdesivir upon V557L mutation. Overall, our studies provided valuable insight into the “template-dependent inhibition mechanism” exerted by Remdesivir on SARS-CoV-2 RdRp and paved venues for an alternative drug design strategy for the treatment of COVID-19.