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

综述：SARS-CoV-2聚合酶和核酸外切酶抑制剂的发现和优化 SARS-CoV-2 RNA依赖性RNA聚合酶（RdRp）（Nsp 12/7/8）和核酸外切酶（ExoN）（Nsp 14/10）发挥作用 在病毒基因组复制和转录中起关键作用。这些酶是高度保守的， 在人类宿主细胞中没有对应物，作为独特的抑制剂靶标。我们发现 SARS-CoV-2 ExoN，作为校对器，去除核苷酸抑制剂，如Remdesivir， 在病毒RNA合成过程中被RdRp掺入病毒RNA。这种机制可能是负责相对 这些药物治疗COVID-19的疗效低下。我们推断，病毒RdRp抑制剂的组合 ExoN可以克服这一缺陷。我们发现RdRp和ExoN的抑制剂协同作用， 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 开发了高分辨率的分子检测方法，以评估SARS-CoV-2 RdRp和ExoN活性， 这些酶的抑制剂我们确定并验证了一套真正的抑制剂，这两种酶。我们 证明口服药物Pibrentasvir，一种HCV NS 5A抑制剂，也抑制SARS-CoV-2 ExoN， Pibrentasvir，以RdRp抑制剂Remdesivir，Favipiravir，Molnupiravir， 和AT-527在很大程度上被保护免于ExoN切除。这些结果表明，所有的核苷/核苷酸- 目前在COVID-19临床试验中的口服候选药物可能会从这种RdRp/ExoN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 将系统地测试阻止SARS-CoV-2复制的能力。我们将进行分子，结构， 体外/体内研究，以优化这种协同策略，开发新的分子作为有效的COVID-19药物。 与MAVDA中心的所有核心密切合作，首先，我们将筛选和表征SARS-CoV-2 RdRp和ExoN抑制剂使用优化的分子测定。第二，我们将进行结构表征 SARS-CoV-2 RdRp和ExoN与已鉴定的抑制剂复合。第三，我们将设计和合成新的 基于上述化合物的分子骨架的SARS-CoV-2 RdRp和ExoN抑制剂， 证明了病毒抑制剂活性。最后，我们将测试SARS-CoV-2 RdRp和ExoN抑制剂的潜力， 使用体外和体内测定的协同作用。这种四管齐下的方法将迭代执行， 口服COVID-19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。

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.