Template-dependent inhibition of coronavirus RNA-dependent RNA polymerase by remdesivir reveals a second mechanism of action

Abstract

Remdesivir (RDV) is a direct-acting antiviral agent that is used to treat patients with severe coronavirus disease 2019 (COVID-19). RDV targets the viral RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have previously shown that incorporation of the active triphosphate form of RDV (RDV-TP) at position i causes delayed chain-termination at position i+3. Here we demonstrate that the S861G mutation in RdRp eliminates chain-termination, which confirms the existence of a steric clash between S861 and the incorporated RDV-TP. With wild type RdRp, increasing concentrations of NTP pools cause a gradual decrease in termination and the resulting read-through increases full-length product formation. Hence, RDV residues could be embedded in copies of the first RNA strand that is later used as a template. We show that the efficiency of incorporation of the complementary UTP opposite template RDV is compromised, providing a second opportunity to inhibit replication. A structural model suggests that RDV, when serving as the template for the incoming UTP, is not properly positioned due to a significant clash with A558. The adjacent V557 is in direct contact with the template base, and the V557L mutation is implicated in low-level resistance to RDV. We further show that the V557L mutation in RdRp lowers the nucleotide concentration required to bypass this template-dependent inhibition. The collective data provide strong evidence to show that template-dependent inhibition of SARS-CoV-2 RdRp by RDV is biologically relevant.

Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

“…Our model provides a structural basis for stalling of RdRp after incorporation of the fourth RMP. This is in agreement with previous studies that have demonstrated that mutations of nsp12(Ser861) reduce the inhibitory effect of remdesivir (Gordon et al, 2020; Tchesnokov et al, 2020). Our structure also shows that the cyano group of each of the four RMP molecules is readily accommodated at the active site, with no apparent steric interactions with the enzyme.…”

“…Initial structural studies with a single copy of RMP in positions −1 (Wang et al, 2020b) and +1 (Yin et al, 2020) demonstrate that RMP is able to be incorporated into the nascent transcript through Watson-Crick base pairing with template strand uracil bases. However, together with previous studies (Gordon et al, 2020; Tchesnokov et al, 2020), our structure and kinetic data show that incorporation of RMP at neither of these positions results in RdRp inhibition. Another recent study (Kokic, 2020) determined multiple structures of RdRp in complex with synthetic substrates in which RMP had been incorporated at specific positions.…”

Screening of potent drug inhibitors against SARS-CoV-2 RNA polymerase: an in silico approach

“…Compound 3j (Remdesivir) is a promising candidate based on existing evidence, and clinical trials of remdesivir are now underway among adults with COVID-19 in hospitals (NIH 2020 ). Remdesivir is a direct-acting antiviral agent used for the treatment of serious 2019 coronavirus disease (COVID-19) patients (Tchesnokov et al 2020 ; Baby et al 2020 ). It showed a mixed outcome with a reasonable side effect in COVID-19 patients (Singh et al 2020 ).…”

The longest persistence of viable SARS-CoV-2 with recurrence of viremia and relapsing symptomatic COVID-19 in an immunocompromised patient – a case study

“…Patient sequences all belonged to clade 20B, corresponding to lineage B.1.1. According to literature data, [18][19][20][21][22][23][24][25] no mutations related to possible resistance to remdesivir were observed, also as minority variant when considering a cut off of 1%.…”