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

• Published in: The FASEB journal Vol. 36 Suppl 1
• Main Authors: Gordon, Calvin J, Lee, Hery W, Tchesnokov, Egor P, Perry, Jason K, Feng, Joy Y, Bilello, John P, Porter, Danielle P, Gotte, Matthias
• Format: Journal Article
• Language: English
• Published: United States 01.05.2022
• ISSN: 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.R4278

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.