Inhibitors of Coronavirus Fidelity and Cap Methylation as Broadly Applicable

2002-2003年SARS-CoV的出现和随后的人际传播，以及2012-2013年中东和欧洲的高毒力人类冠状病毒HCoV-EMC的出现，都证明了CoV的移动潜力和传播性，并强调了对广泛有效治疗方法的迫切和关键需求。项目2的总体目标是确定两个高度保守的冠状病毒过程的抑制剂，复制保真度和RNA加帽，这对SARS冠状病毒的毒力和体内存活至关重要。多种病毒蛋白和酶活性对这些过程至关重要，包括CoV 3 '-to-5'核糖核酸外切酶（保真度; nsp 14-ExoN）和2 '-O-甲基转移酶（加帽; nsp 16 - 0 MTase）活性。与这些过程的重要性一致，我们已经表明，通过ExoN或OMTase的基因失活，分别降低复制保真度和消融RNA加帽，导致在体内被深度减毒的有复制能力的病毒。在目标1和2中，我们将与筛选核心（核心B）和药物化学领导开发核心（核心C）合作，以鉴定、表征和优化SARS-CoV保真度和RNA加帽的小分子抑制剂。一旦确定了活性化合物，我们将确定它们的作用机制，测试病毒耐药性的发展，并确定它们在CoV家族中的活性。在目标S中，我们将与核心C合作，在SARSCoV疾病严重程度的渐进分层模型中化学优化和测试先导化合物的体内功效，并评估体内耐药性的发展。丹尼森和巴里克实验室的互补专业知识，广泛的初步数据集，最先进的技术，以及SR在药物化学，高通量筛选和药物开发领域的专业知识，将为成功识别，确认和体内测试先导化合物做出重大贡献。最终，抑制体内发病所需的这两种保守且不同的途径将允许治疗地方性和新出现的CoV，并可能减少病毒耐药性的出现。

Both the emergence and subsequent human-to-human transmission of SARS-CoV in 2002-2003, and ofthe highly virulent human coronavirus HCoV-EMC in the Middle East and Europe in 2012-2013 exemplifies CoV movement potential and transmissibility, and underscores the urgent and critical need for a broadly efficacious therapeutics. The overall goal of Project 2 is to identify inhibitors of two highly conserved CoV processes, replication fidelity and RNA capping, that are essential for SARS-CoV virulence and survival in vivo. Multiple viral proteins and enzymatic activities are critical for these processes, including CoV 3'-to-5' exoribonuclease (fidelity; nsp14-ExoN) and 2'-0-methyltransferase (capping; nsp16-0MTase) activities. Consistent with the importance of these processes, we have shown that decreased replication fidelity and ablation of RNA capping through genetic inactivation of either ExoN or OMTase, respectively, results in replication competent viruses that are profoundly attenuated in vivo. In Aims 1 and 2, we will work with the Screening Core (Core B) and the Medicinal Chemistry lead Development Core (Core C) to identify, characterize, and optimize small molecule inhibitors of SARS-CoV fidelity and RNA capping. Once active compounds are identified, we will define their mechanism of action, test for the development of virus resistance, and determine their activity across the CoV family. In Aim S, we will work with Core C to chemically optimize and test the in vivo efficacy of lead compounds in progressively tiered models of SARSCoV disease severity, and assess the development of drug resistance in vivo. The complementary expertise ofthe Denison and Baric Labs, extensive preliminary datasets, state-of-the-art technologies, and the expertise of SR in the areas of medicinal chemistry, high-throughput screening, and drug development will contribute significantly to the successful identification, confirmation, and in vivo testing of lead compounds. Ultimately, inhibiting these two conserved and distinct pathways required for in vivo pathogenesis will allow for the treatment of endemic and emerging CoVs and potentially reduce the emergence of viral resistance.