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 additional 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 with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3’-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2’-O-methyl UTP), and 3 did not terminate the polymerase reaction (2’-fluoro-dUTP, 2’-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2’ -OH group to excise mismatched bases at the 3’-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2’-OH, have a blocked 2’-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.

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冠状病毒聚合酶催化SARS和COVID-19的核苷酸类似物终止RNA合成的文库

SARS-CoV-2是冠状病毒家族的成员，对当前的全球COVID-19大流行负责。我们先前证明了五个核苷酸类似物可抑制SARS-CoV-2 RNA依赖性RNA聚合酶（RdRp），包括Sofosbuvir，Alovudine，Zidovudine，Tenofovir alafenamide和Emtricitabine的活性三磷酸形式。我们在这里报告对具有多种结构和化学特征作为SARS-CoV和SARS-CoV-2的RdRps抑制剂的其他三磷酸核苷类似物文库的评估。这些特征包括糖上的修饰（2'或3'修饰，碳环，无环或双脱氧核苷酸）或碱基上的修饰。目的是确定不仅终止这些冠状病毒RdRps催化的RNA合成的核苷酸类似物，但也有可能抵抗病毒的核酸外切酶活性。我们就这些核苷酸类似物在聚合酶反应中被RdRps掺入并防止进一步掺入的能力进行了研究。尽管所有测试的11个分子均显示出结合，但6个分子显示聚合酶反应立即终止（卡波韦尔三磷酸，更昔洛韦三磷酸，司他夫定三磷酸，恩替卡韦三磷酸，3'-O-甲基UTP和Biotin-16-dUTP），其中2个显示延迟终止（西多福韦二磷酸和2'-O-甲基UTP）和3并未终止聚合酶反应（2'-氟-dUTP，2'-氨基-dUTP和Desthiobiotin-16-UTP）。冠状病毒基因组编码一个核酸外切酶，该酶显然需要一个2'-OH基团才能切除3'-末端错配的碱基。在这个研究中，我们评估的所有核苷三磷酸类似物均形成沃森-克里克样碱基对。所有显示出终止的核苷酸类似物要么缺少2'-OH，要么被封闭的2'-OH，或显示延迟的终止。因此，这些核苷酸可能具有抗核酸外切酶活性的潜力，这是我们将来将要研究的特性。此外，其中五个核苷酸类似物（溴西多福韦/西多福韦，阿巴卡韦，缬更昔洛韦/更昔洛韦，司他夫定和恩替卡韦）的前药已获FDA批准用于其他病毒感染，其安全性是众所周知的。因此，可以快速评估它们作为COVID-19的潜在疗法。因此，这些核苷酸可能具有抗核酸外切酶活性的潜力，这是我们将来将要研究的特性。此外，其中五个核苷酸类似物（溴西多福韦/西多福韦，阿巴卡韦，缬更昔洛韦/更昔洛韦，司他夫定和恩替卡韦）的前药已获FDA批准用于其他病毒感染，其安全性是众所周知的。因此，可以快速评估它们作为COVID-19的潜在疗法。因此，这些核苷酸可能具有抗核酸外切酶活性的潜力，这是我们将来将要研究的特性。此外，其中五个核苷酸类似物（溴西多福韦/西多福韦，阿巴卡韦，缬更昔洛韦/更昔洛韦，司他夫定和恩替卡韦）的前药已获FDA批准用于其他病毒感染，其安全性是众所周知的。因此，可以快速评估它们作为COVID-19的潜在疗法。