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'-NH₂-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.

Keywords: COVID-19; Exonuclease; Nucleotide analogues; RNA-Dependent RNA polymerase; SARS-CoV-2.

Fig. 1

Chemical structures of nucleoside triphosphate analogues used in this study.

Fig. 2

Structures of viral nucleoside/nucleotide inhibitors, example prodrugs and active triphosphate forms. The compounds Ganciclovir, Abacavir, Cidofovir, Stavudine and Entecavir (left), example prodrug forms (middle) and their active triphosphate forms (right).

Fig. 3

Incorporation of 2′-OMe-UTP, Sta-TP and Biotin-dUTP by SARS-CoV-2 RdRp to terminate the polymerase reaction. The sequences of the primer and template used for this extension reaction, 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 2′-OMe-UTP (a), Sta-TP (b) and Biotin-dUTP (c) with pre-assembled 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 detailed procedure is shown in the Materials and Methods section. The accuracy for m/z determination is ±10 Da.

Fig. 4

Incorporation of Cid-DP by SARS-CoV-2 RdRp to achieve delayed termination of the polymerase reaction. The sequences of the primer and template used for this extension reaction are shown at the top of the figure. The polymerase extension reaction was performed by incubating Cid-DP, UTP and ATP with pre-assembled 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.

Fig. 5

Incorporation of Car-TP, Ent-TP and Gan-TP by SARS-CoV-2 RdRp to terminate the polymerase reaction. The sequences of the primer and template used for this extension reaction are shown at the top of the figure. Polymerase extension reactions were performed by incubating Car-TP, UTP, ATP and CTP (a), Ent-TP, UTP, ATP and CTP (b), and Gan-TP, UTP, ATP and CTP (c) with pre-assembled 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.