Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase
Authors: Xin-Jie Chu
Sheng Ding
Lan Hua
Wen Xiong
Lan Xu
Weinan Zheng
Jialei Sun
Gaofei Lu
Xi Zhang
Journal: Antimicrobial Agents and Chemotherapy (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: RNA-dependent RNA polymerase; SARS-CoV-2; discrimination value; favipiravir; nsp12-nsp8-nsp7
Affiliations: Global Health Drug Discovery Institute, Beijing, China  ., Global Health Drug Discovery Institute, Beijing, China., School of Pharmaceutical Sciences, Tsinghua University, Beijing, China.

Abstract:  Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). 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 analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. 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 for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs. 

Retracted: No

PubPeer comments:
 Fig1: Why co-purified NSP7-8 are in equal amounts on the gel? Since NSP7, 8 and 12 are in equimolar (one molecule each) then the amount of protein on SDS gel will be different. NSP8 amount will be almost twice of NSP7. Fig2C, lane 2-8: Once co-purified, how can authors determine the amount (micromolar unit?) of each separately? Here the ratio of NSP7 to 8 is ~5. Method section:  nsp7 and nsp8 protein expression and purification : it appears that both NSP8 and 7 were cloned in pMAL by replacing MBP e.g. MBP was replaced by NSP8 in pMAL then why authors write-  SARS-CoV-2 **nsp8 gene** (nucleotides 12092 to 12685; strain name, Wuhan-Hu-1; GenBank no. MN908947.3) was synthesized de novo by GenScript (Nanjing, China) and cloned into a pMal-c5X vector under tac promoter control (without a maltose-binding protein [MBP] sequence). Specifically, the entire MBP sequence of pMal-c5X (nucleotides 1527 to 2628) was replaced with **the nsp7 gene **sequence with the addition of an N-terminal 6-His tag . Is NSP7 in the above paragraph is a typo? If both NSP7 and NSP8 were individually His-tagged then how can they be  co-purified ? Thanks , 
 #1 Why co-purified NSP7-8 are in equal amounts on the gel? answer: They are not in equal amounts. Nsp7 and nsp8 concentration in  co-purified nsp7-8 was quantified from the protein gel stained with Coomassie blue using known concentration of BSA as a standard. Thanks for pointing out. NSP7 in the above paragraph was a typo, and it should be NSP8. ,