A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19

Abstract

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2′ or 3′ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3′-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2′-OMe-UTP), and 3 did not terminate the polymerase reaction (2′-F-dUTP, 2′-NH 2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2′-OH at the 3′-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2′-OH, have a blocked 2′-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.

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

“… 11 We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp. 18 …”

“… 29 , 30 We previously studied Car-TP as an inhibitor of the SARS-CoV and SARS-CoV-2 RdRp using a higher concentration than in the current study. 18 …”

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

“…Potential inhibitors have been investigated to target various steps in the Coronavirus infectious cycle, including the viral replication machinery. 10 However, as of now, no effective therapeutic is available to treat serious coronavirus infections such as COVID- 19. The RdRp is one of the key targets for antiviral drug development.…”

“…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. 20 Jácome et al recently recommended Sofosbuvir as a possible antiviral for COVID-19, based on structural studies and bioinformatic analysis.…”

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].…”