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Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.

ABSTRACT: COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp). However, the enzymatic mechanism and efficiency of Remdesivir have not been determined, and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in E. coli, followed by purification and kinetic analysis of an untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast (k _cat = 240-680 s^-1) and processive (k _off = 0.013 s^-1) RNA polymerization. The specificity constant (k _cat /K _m ) for Remdesivir triphosphate (RTP) incorporation (1.29 μM^-1s^-1) is higher than that for the competing ATP (0.74 μM^-1 s^-1). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the standard for development of informative enzyme assays to screen for new inhibitors.

SUBMITTER: Dangerfield TL

PROVIDER: S-EPMC7695572 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Remdesivir (GS-5734) is a 1'-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1'-cyano modification. Compounds with modifications at the 2'-position show different patterns of inhibition. While 2'-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

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Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.

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