658.6 - Efficient Incorporation and Template-Dependent Polymerase Inhibition are Major Determinants for the Broad-Spectrum Antiviral Activity of Remdesivir

Calvin Gordon (Department of Medical Microbiology and Immunology, University of Alberta), Hery Lee (University of Alberta), Egor Tchesnokov (University of Alberta), Jason Perry (Gilead Science, Inc.), Joy Feng (Gilead Science, Inc.), John Bilello (Gilead Science, Inc.), Danielle Porter (Gilead Science, Inc.), Matthias Gotte (University of Alberta)

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, e.g. SARS-CoV-2 and hepatitis C virus (HCV) and non-segmented negative-sense RNA viruses, e.g. Nipah virus (NiV), while segmented negative-sense RNA viruses such as influenza (Flu) virus or Crimean-Congo hemorrhagic fever virus (CCHFV) are not sensitive to the drug. The reasons for this apparent pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases (RdRp) and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. The results of this study revealed a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions  is heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP is seen with all polymerases. Molecular modeling suggests a steric conflict between the 1’-cyano group of RDV-MP and conserved residues of RdRp motif F. We conclude that future efforts in the development of nucleotide analogues with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1’-modification.

C.J.G, H.W.L, and E.P.T contributed equally to this work

This study was supported by grants to MG from the Canadian Institutes of Health Research (CIHR, grant number 170343), Gilead Sciences and from the Alberta Ministry of Economic Development, Trade and Tourism by the Major Innovation Fund Program for the AMR amp;ndash; One Health Consortium.