Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease

SARS-CoV-2聚合酶和核酸外切酶抑制剂的发现与优化 SARS-CoV-2依赖RNA聚合酶(RdRp)(Nsp12/7/8)和外显子(Nsp14/10)的作用 在病毒基因组复制和转录中的关键作用。这些酶在 冠状病毒，并且在人类宿主细胞中没有对应的基因，作为独特的抑制物靶标。我们发现 SARS-CoV-2外显子起校对作用，移除核苷酸抑制物，如Remdesivir，这些抑制物是 RdRp在合成过程中被RdRp掺入病毒RNA。这一机制可能负责相对 这些药物治疗新冠肺炎的疗效较低。我们推断，病毒RdRp的抑制剂组合 而外显子可以克服这一缺陷。我们发现RdRp和外显子的抑制剂具有协同作用。 克服单独使用RdRp抑制剂在体外阻断SARS-CoV-2复制的不足。我们有 建立高分辨分子检测SARS-CoV-2 RdRp和外显子活性的方法 这些酶的抑制剂。我们鉴定并验证了一组针对这两种酶的真正的抑制剂。我们 证明了口服药物匹布列韦，一种丙型肝炎病毒NS5A抑制剂，也抑制SARS-CoV-2外显子，并且在 Pibrentasvir的存在，以RdRp抑制剂Remdesivir，Favipiravir，Molnupiravir， 和AT-527在很大程度上没有被外显子切除。这些结果表明，所有的核苷/核苷酸- 目前正在进行新冠肺炎临床试验的口服候选药物可能会从这个RdRp/外显子中受益 抑制剂组合方法。 在这个项目中，我们将专注于发现和优化这两种酶的新抑制剂。他们的 将对阻止SARS-CoV-2复制的能力进行系统测试。我们将执行分子、结构和 体外和体内研究旨在优化这一协同策略，以开发新的分子作为有效的新冠肺炎药物。 在与MAVDA中心所有核心的密切合作下，首先，我们将对SARS-CoV-2进行筛查和特征分析 RdRp和外显子抑制剂使用优化的分子分析。第二，我们将执行结构表征 SARS-CoV-2的RdRp和外显子与已鉴定的抑制物形成络合。第三，我们将设计和合成新的 基于上述化合物的分子支架的SARS-CoV-2 RdRp和外显子抑制剂 显示出病毒抑制活性。最后，我们将测试SARS-CoV-2 RdRp和外显子抑制剂的潜力 采用体外和体内试验的协同效应。这个四管齐下的方法将迭代执行，用于 口腔新冠肺炎疗法的发展。我们期待新开发的SARS-CoV-2 RdRp抑制剂 将抑制黄病毒(ZIKV，DENV)和甲病毒(CHIKV，EEEV)中的RNA聚合酶，我们将评估 它们抑制这些病毒的能力也是如此。

Summary: Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (Nsp12/7/8) and exonuclease (ExoN) (Nsp14/10) play critical roles in viral genome replication and transcription. These enzymes are highly conserved among the coronaviruses and have no counterparts in human host cells, serving as unique inhibitor targets. We discovered that SARS-CoV-2 ExoN, acting as a proofreader, removes nucleotide inhibitors, such as Remdesivir, that are incorporated by RdRp into viral RNA during its synthesis. This mechanism might be responsible for the relatively low efficacy of these drugs for treating COVID-19. We reasoned that combinations of inhibitors of the viral RdRp and ExoN could overcome this deficiency. We showed that inhibitors of the RdRp and ExoN act synergistically to overcome deficiencies of using RdRp inhibitors alone to block SARS-CoV-2 replication in vitro. We have developed high resolution molecular assays to assess SARS-CoV-2 RdRp and ExoN activities to screen inhibitors of these enzymes. We identified and validated a set of bona fide inhibitors for both enzymes. We demonstrated that the oral drug Pibrentasvir, an HCV NS5A inhibitor, also inhibits SARS-CoV-2 ExoN, and in the presence of Pibrentasvir, RNAs terminated with the RdRp inhibitors Remdesivir, Favipiravir, Molnupiravir, and AT-527 were largely protected from ExoN excision. These results indicate that all the nucleoside/nucleotide- based oral drug candidates currently in COVID-19 clinical trials will potentially benefit from this RdRp/ExoN inhibitor combination approach. In this project, we will focus on the discovery and optimization of new inhibitors of these two enzymes. Their ability to stop SARS-CoV-2 replication will be systematically tested. We will perform molecular, structural, and in vitro/in vivo studies to optimize this synergistic strategy to develop new molecules as effective COVID-19 drugs. In close collaboration with all the Cores of the MAVDA Center, first, we’ll screen and characterize SARS-CoV-2 RdRp and ExoN inhibitors using optimized molecular assays. Second, we’ll perform structural characterization of SARS-CoV-2 RdRp and ExoN complexed with the identified inhibitors. Third, we’ll design and synthesize new SARS-CoV-2 RdRp and ExoN inhibitors based on the molecular scaffolds of the above compounds that have demonstrated viral inhibitor activity. Finally, we’ll test SARS-CoV-2 RdRp and ExoN inhibitors for their potential synergistic effects using in vitro and in vivo assays. This four-pronged approach will be performed iteratively for the development of oral COVID-19 therapeutics. We expect the newly developed SARS-CoV-2 RdRp inhibitors will inhibit RNA polymerases in flaviviruses (ZIKV, DENV) and alphaviruses (CHIKV, EEEV), and we will assess their ability to inhibit these viruses as well.