Project 4: Nuclease Inhibitors for Viruses of Pandemic Concern

项目4 -大流行病毒的核酸酶抑制剂 摘要 SARS-CoV-2（SARS 2）和高致病性沙粒病毒，包括拉沙病毒（LASV）、朱宁病毒和 马丘波病毒共享一个结构和功能相关的3 '至5'核糖核酸外切酶（ExoN）结构域， 在由易错病毒RNA依赖性RNA聚合酶进行RNA合成过程中校对的重要作用 （RdRp）和抑制宿主抗病毒应答。项目4正在寻求化学抑制ExoN从这些 大流行关注的病毒，目的是通过致命诱变阻断病毒复制， 通过重新激活宿主的干扰素反应来引起病毒发病。我们的团队为 这些病毒ExoN酶的结构和功能的理解，包括阐明其第一个原子 催化机理的结构和表征。我们还开发了基于荧光的 定量分析ExoN催化活性的测定，包括一种以荧光RNA为特征的新型测定 适体底物，其能够在超高通量筛选（uHTS）中进行功能获得性读出。 此外，我们使用DNA编码化学技术（DEC-Tec）来获得选择性结合剂， SARS 2 ExoN和含有ExoN结构域的LASV核蛋白（NP）。基于这些先前和初步的 研究，我们将继续与核心B密切合作，并使用3种互补方法（uHTS，DEC-Tec， 和虚拟筛选）以鉴定一流的病毒ExoN抑制剂。然后我们将利用 核心C和核心D分别在药物化学和结构生物学，以提高效力， 选择性和药效学/动力学特性，用于细胞中的详细抗病毒研究， 动物模型的核心E。这些研究将提供具有独特作用机制的抗病毒候选药物 以补充针对包括RdRp在内的已确定的抗病毒靶点开发的药物， 迫切需要针对SARS 2和高致病性沙粒病毒的新型抗病毒药物， 致命的出血热感染。

Project 4 – Nuclease Inhibitors for Viruses of Pandemic Concern Abstract SARS-CoV-2 (SARS2) and highly pathogenic arenaviruses including Lassa virus (LASV), Junin virus, and Machupo virus share a structurally and functionally related 3'-to-5' exoribonuclease (ExoN) domain, which plays essential roles in proofreading during RNA syntheses by the error-prone viral RNA-dependent RNA polymerase (RdRp) and suppressing host antiviral responses. Project 4 is pursuing chemical inhibition of ExoN from these viruses of pandemic concern, with the goal of blocking viral replication by lethal mutagenesis as well as mitigating viral pathogenesis by reactivating host’s interferon responses. Our team has contributed extensively to the structural and functional understanding of these viral ExoN enzymes, including the elucidation of their first atomic structures and characterization of catalytic mechanisms. We have also developed robust fluorescence-based assays to quantitatively analyze the ExoN catalytic activities, including a novel assay featuring fluorogenic RNA aptamer substrates that enables a gain-of-function readout in ultra-high-throughput screening (uHTS). Furthermore, we have used DNA-encoded chemistry technology (DEC-Tec) to obtain selective binders to SARS2 ExoN and LASV nucleoprotein (NP) containing the ExoN domain. Building on these prior and preliminary studies, we will continue to work closely with Core B and use 3 complementary approaches (uHTS, DEC-Tec, and Virtual screening) to identify first-in-class viral ExoN inhibitors. We will then leverage the deep expertise of Core C and Core D in medicinal chemistry and structural biology, respectively, to enhance the potency, selectivity, and pharmacodynamic/kinetic properties of hit compounds for detailed antiviral studies in cell and animal models by Core E. These studies will deliver antiviral drug candidates with a distinct mechanism of action to complement those developed against established antiviral targets including RdRp, helping to address the critical need for novel antiviral drugs against both SARS2 and the highly pathogenic arenaviruses that cause fatal hemorrhagic fever infections.