Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19

Abstract

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

Keywords: COVID-19; RNA-dependent RNA polymerase; SARS-CoV-2; nucleotide analogues.

Figure 1

Structures of four prodrug viral inhibitors. Top: Prodrug (phosphoramidate) form; Bottom: Active triphosphate form.

Figure 2

Structures of three viral inhibitors. Top: Nucleoside form; Bottom: Active triphosphate form.

Figure 3

Incorporation of 2′-F,Me-UTP, 3′-F-dTTP, TFV-DP, and 3′-N₃-dTTP by SARS-CoV-2 RdRp to terminate the polymerase reaction. The sequences of the primer and template used for these extension reactions, which are at the 3′ end of the SARS-CoV-2 genome, are shown at the top of the figure. Polymerase extension reactions were performed by incubating (a) 2′-F,Me-UTP, (b) 3′-F-dTTP, (c) UTP + TFV-DP, and (d) 3′-N₃-dTTP with preassembled SARS-CoV-2 polymerase (nsp12, nsp7, and nsp8), the indicated RNA template and primer, and the appropriate reaction buffer, followed by detection of reaction products by MALDI-TOF MS. The accuracy for m/z determination is ±10 Da.

Figure 4

Incorporation of TFV-DP and Car-TP by SARS-CoV-2 RdRp to terminate the polymerase reaction. The sequences of the primers and template used for these extension reactions, which are at the 3′ end of the SARS-CoV-2 genome, are shown at the top of the figure. Polymerase extension reactions were performed by incubating (a) TFV-DP and (b) UTP + ATP + CTP + Car-TP with preassembled SARS-CoV-2 polymerase (nsp12, nsp7, and nsp8), the indicated RNA template and primers, and the appropriate reaction buffer, followed by detection of reaction products by MALDI-TOF MS. The accuracy for m/z determination is ±10 Da.

Figure 5

Incorporation of Lam-TP and Ec-TP by SARS-CoV-2 RdRp catalyzed reaction. The sequences of the primer and template used for these extension reactions, which are at the 3′ end of the SARS-CoV-2 genome, are shown at the top of the figure. Polymerase extension reactions were performed by incubating (a) UTP + ATP + Lam-TP and (b) UTP + ATP + Ec-TP with preassembled SARS-CoV-2 polymerase (nsp12, nsp7, and nsp8), the indicated RNA template and primer, and the appropriate reaction buffer, followed by detection of reaction products by MALDI-TOF MS. The accuracy for m/z determination is ±10 Da.