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.

Sofosbuvir Terminated RNA is More Resistant to SARS-CoV-2 Proofreader than RNA Terminated by Remdesivir

“…The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, shown to be required for the processive polymerase activity of nsp12, were cloned and purified as described. 3 The 3'exonuclease, referred to as nsp14, and its protein cofactor, nsp10, were purchased from LSBio (Seattle, WA). Sofosbuvir triphosphate (PSI-7409) was purchased from Sierra Bioresearch (Tucson, AZ).…”

“…18 We subsequently demonstrated that Sofosbuvir triphosphate is incorporated by the relatively low fidelity SARS-CoV and SARS-CoV-2 RdRps, serving as an immediate polymerase reaction terminator, but not by a host-like high fidelity DNA polymerase. 2,3 We also reported that a library of additional nucleotide analogues with a variety of structural and chemical features terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19. 19 Gordon et al performed a kinetic study, including the determination of Km values for triphosphates of Remdesivir, Sofosbuvir and other nucleotide analogues.…”

Functional and druggability analysis of the SARS-CoV-2 proteome

“…Favipiravir (also called avifavir), used in Hepatitis C Virus (HCV) treatment, is also a promising drug ( Wang et al, 2020a ). Other antivirals, such as sofosbuvir, alovudine, tenofovir alafenamide, AZT, abacavir, lamivudine, emtricitabine, carbovir, ganciclovir, stavudine, and entecavir, are also incorporated by SARS-CoV-2 RdRp and block replication in vitro ( Chien et al, 2020 ). Another type of broad spectrum ribonucleoside analog, β-D-N4-hydroxycytidine (EIDD-1931), has been shown to inhibit SARS-CoV-2, SARS-CoV and MERS-CoV in cell culture by increasing the mutation transition rate, probably exceeding the proofreading ability granted by the ExoN ( Sheahan et al, 2020 ).…”

Functional and druggability analysis of the SARS-CoV-2 proteome

“…Favipiravir (also called avifavir), used in Hepatitis C Virus (HCV) treatment, are also promising drugs [93]. Nevertheless, other antivirals, such as sofosbuvir, alovudine, tenofovir alafenamide, AZT, abacavir, lamivudine, emtricitabine, carbovir, ganciclovir, stavudine, and entecavir, are incorporated by SARS-CoV-2 RdRp and block replication in vitro [95,96]. Another type of broad spectrum ribonucleoside analog, β-D-N4-hydroxycytidine (EIDD-1931), has been shown to inhibit SARS-CoV-2, SARS-CoV and MERS-CoV in cell culture, by increasing the mutation transition rate, probably exceeding the proofreading ability granted by ExoN [97].…”