Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase

Abstract

Nucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in literature, we found that the incorporation efficiency of remdesivir-TP is higher than ATP, and incorporation of remdesivir-TP caused delayed chain termination which can be overcome by higher concentrations of the following nucleotide to be incorporated. Our data also showed that the delay chain termination pattern caused by remdesivir-TP incorporation is different for different template sequence. Multiple incorporations of remdesivir-TP caused chain termination in our assay condition. Incorporation of sofosbuvir-TP is very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

Potential Novel Thioether-Amide or Guanidine-Linker Class of SARS-CoV-2 Virus RNA-Dependent RNA Polymerase Inhibitors Identified by High-Throughput Virtual Screening Coupled to Free-Energy Calculations

“…This essential viral enzyme is highly conserved among (+ssRNA) viral species and, as such, devoid of a human counterpart promoting this protein to a viable, under-investigated and prospective therapeutic target [ 32 , 33 ]. Moreover, the complexity of RNA replication machinery involving a synchronized action of multiple viral and host proteins towards final RNA polymerization makes this target a difficult system for designing in vitro biological experiments [ 34 , 35 ]. Furthermore, current postulated actives on RdRp rely on (infected) whole cell-line experimental data and on conducted preclinical in vivo data, further emphasizing the need for a thorough in silico computer-assisted drug design approach [ 36 , 37 ].…”

Targeting SARS-CoV-2 Polymerase with New Nucleoside Analogues

“…In all these, the polymerization of RNA is checked with the use of the catalytic subunit and appropriate nucleotides [ 44 ]. In our study we used an alternative of primer extension assay as described by Lu et al [ 45 ]. The RNA template is polymerized using the RNA-dependent-RNA polymerase, when non-modified analogues are used, while the polymerization is blocked when modified nucleotides are incorporated into the reaction.…”

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19

“…AS2 has been shown to transfer all natural NMPs, and some analogs, to target proteins [20,21], implying that mis-NMPylation may happen, possibly with deleterious consequences for viral replication. To act as an inhibitor, an NA must outcompete natural NTPs; indeed, remdesivir triphosphate (RDV-TP) outcompetes ATP in different in vitro RNA synthesis assays [44][45][46] and AT-527 triphosphate (a.k.a. AT-9010), a 2 -fluoro-2 -C-methyl GTP, has been reported to be an efficient competitor of GTP in both active sites [12].…”