Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

Abstract

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 efficacy 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. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1’-cyano group of the inhibitor and residues of the structurally conserved 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.

Mutations in the SARS-CoV-2 RNA-dependent RNA polymerase confer resistance to remdesivir by distinct mechanisms

“…However, increases in nucleoside-triphosphate (NTP) concentrations can overcome this obstacle and RNA synthesis can continue, resulting in RNA strands with incorporated RDV-monophosphate (RDV-MP) residues. In this setting, template-dependent inhibition of RNA synthesis occurs because of compromised incorporation of the complementary uridine-triphosphate (UTP) opposite RDV-MP ( 12 – 14 ).…”

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants

“…At present, a structure of the preincorporated state of RDV-TP in the RdRp active site is still unavailable. We built a model, described previously ( 14 , 15 , 36 ), based on an existing structure of the polymerase complex, Nsp12/(Nsp8) 2 /Nsp7/(Nsp13) 2 , with primer and template RNA (PDB no. 6XEZ ) ( 37 ).…”

The Nucleoside/Nucleotide Analogs Tenofovir and Emtricitabine Are Inactive against SARS-CoV-2

“…It was previously shown that the RdRp of SARS-CoV-2 incorporated the pharmacologically active RDV triphosphate (RDV-TP) species approximately three-fold more efficiently than its natural counterpart ATP [14]. Moreover, the biochemical evaluation of viral RdRp from different RNA viruses demonstrated efficient RDV-TP incorporation is required to elicit an antiviral effect [15]. To address the differences observed in the antiviral activity of TFV-DP and FTC-TP against SARS-CoV-2, we determined the efficiency at which the NRTIs were incorporated by SARS-CoV-2 RdRp (Table 3).…”

“…The pFastBac-1 (Invitrogen, Burlington, ON, Canada) plasmid with the codon-optimized synthetic DNA sequences (GenScript, Piscataway, NJ, USA) coding for SARS-CoV-2 (NCBI: QHD43415.1) nsp5, nsp7, nsp8, and nsp12 were used as a starting material for protein expression in insect cells (Sf9, Invitrogen, Waltham, MA, USA) [14,15]. We employed the MultiBac (Geneva Biotech, Indianapolis, IN, USA) system for protein expression in insect cells (Sf9, Invitrogen) according to published protocols [30,31].…”