Remdesivir is Effective in Combating COVID-19 Because it is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase

Abstract

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp) complex. However, the enzymatic mechanism and efficiency of Remdesivir have not been determined and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in E. coli, purification, and kinetic analysis of the untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast (kcat = 200—500 s-1) and processive (koff = 0.013 s-1) RNA polymerization and that the specificity constant (kcat/Km) for Remdesivir triphosphate (RTP) incorporation (1.3 μM-1s-1) is almost twice that for the competing ATP (0.7 µM-1 s-1). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the stage for development of informative enzyme assays to screen for new inhibitors.Funding: This work was supported by grants from NIAID (1R01AI110577 to KAJ) and the Welch Foundation (F-1604 to KAJ). Remdesivir triphosphate was a gift kindly provided by Brian Schultz and Joy Feng at Gilead Sciences.Conflict of Interest: K.A.J. is president of KinTek Corporation, which donated the quench flow instruments and KinTek Explorer software used in these studies.