Mechanism of Inhibition of the Reproduction of SARS-CoV-2 and Ebola Viruses by Remdesivir

Abstract

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro , and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.

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

“…Efficient selective incorporation of RDV-TP, calculated as ratio of efficiency of incorporation of ATP over RDV-TP, is seen with SARS-CoV-2 (0.3), HCV (0.9), EBOV (4.0), NiV (1.6) and RSV enzymes (2.7). In contrast, much higher selectivity values for LASV ( 20 ), CCHFV ( 41 ), and FluB (68) enzymes suggest poor RDV-TP substrate usage. The ability of a nucleotide analogue inhibitor to be incorporated by viral RdRp enzymes is largely dependent on variations in the residues that define the nucleotide binding site ( 44 ).…”

“…For SARS-CoV-2 RdRp, it has been demonstrated that higher NTP concentrations can overcome inhibition at "i+3" ( 26 , 27 , 36 , 37 , 38 , 39 ). Others have therefore used a different terminology and refer to "delayed translocation" ( 39 ), “delayed chain extension” ( 39 ), “delayed stalling" ( 36 ), “delayed intervention" ( 40 ), or enzyme "pausing" ( 27 , 37 , 40 , 41 ). “Pausing” has also been observed on long templates that better mimic the RNA genome ( 37 ).…”

Comprehensive Consensus Analysis of SARS-CoV-2 Drug Repurposing Campaigns

“…RdRp shares significant homology within the RNA virus Coronaviridae family and is a popular target for SARS-CoV-2 repositioning studies due to a given drug’s prospect of high selectivity and low probability of associated cytotoxicity. , Many nucleoside analogues have been proposed due to low RdRp replication fidelity . Due to the inhibitory potential of remdesivir for the RdRp in Ebola virus, , several virtual screening campaigns were conducted. − , Despite the lack of consensus for hits identified among the virtual repurposing campaigns, two of the in silico shortlisted drugs were reported among the hits identified in cell-based repurposing studies, namely, digoxin (a cardiac glycoside; EC 50 = 0.07 μM) and ritonavir (an HIV protease inhibitor; AC 50 = 22.53 μM). ,,, …”

“…82,83 Many nucleoside analogues have been proposed due to low RdRp replication fidelity. 85 Due to the inhibitory potential of remdesivir for the RdRp in Ebola virus, 67,86 several virtual screening campaigns were conducted. [83][84][85]87 Despite the lack of consensus for hits identified among the virtual repurposing campaigns, two of the in silico shortlisted drugs were reported among the hits identified in cell-based repurposing studies, namely, digoxin (a cardiac glycoside; EC 50 = 0.07 μM) 20 and ritonavir (an HIV protease inhibitor; AC 50 = 22.53 μM).…”

Structural Insights into Binding of Remdesivir Triphosphate within the Replication–Transcription Complex of SARS-CoV-2

“…Nevertheless, RMP-containing viral RNAs are likely nonfunctional and thus are partially responsible for the suppression of viral reproduction. 13 In fact, the incorporation of RDV and its effects on viral RNA production in the SARS-CoV-2 infected cell lines is highly strand dependent. 12 Therefore, a new class of anti-SARS-CoV-2 inhibitors could be rationally designed, including the formation of dead-end inhibitory ternary complexes at the pol active site, translocation-inhibited complexes, or exoribonuclease-inhibited complexes.…”