Development of Novel Antivirals Targeting Viral RNA Methylation

项目4：开发针对病毒RNA甲基化的新型抗病毒药物 摘要 冠状病毒通过两个甲基转移酶(MTase)：Nsp14的协调作用来限制它们的RNA，Nsp14催化 病毒RNA 5‘端GTP的N7-胍甲基化，以及形成C2’-O-甲基的Nsp16。 核糖腺嘌呤位于随后的核苷酸。通过模仿宿主细胞的信使核糖核酸，得到的帽子结构是 对于免疫逃避、稳定病毒RNA和高效翻译至关重要。而功能突变的丢失 在Nsp14的MTase结构域对SARS-CoV-2致死，携带突变Nsp16的SARS-CoV毒株具有低的 毒力，这表明靶向这些酶，无论是单独的还是与其他病毒蛋白结合， 有很强的治疗潜力。在这项应用中，我们建议开发针对MTase的抗病毒药物 Nsp14和Nsp16的活动。我们的方法将评估RNA封端MTase作为一个新靶点的潜力 开发抗病毒药物的家庭。重要的是，因为这两种酶在冠状病毒中都是保守的 已知会感染人类，这种方法可能为泛冠状病毒作用的发展提供足迹 探员们。 每种MTase的抑制剂将使用小分子发现方法的组合来确定： 计算对接、片段链接和合并以及高通量筛选(HTS)，以识别新的 化学类型。考虑到按需制作图书馆的最新发展，我们建议 使用超大型文库对接来确定候选抑制剂。药物类化合物在内部的供应情况 小分子文库将有助于通过高温超导鉴定缓蚀剂。热门化合物将进行一系列测试 活性分析，并使用直接结合策略进行验证。实验确定的MTase的结构 在含有小分子抑制剂的复合体中将用于指导优化，辅以获得按需制造 图书馆。已确定的抑制剂将根据其相对于一个综合小组的选择性进行优先排序 SARS-CoV-2感染细胞模型中的人MTase和抗病毒活性。后来的药物 在动物模型中的化学优化和抗病毒活性的评估有望导致优化 先导化合物，这将由我们的行业合作伙伴(罗氏)进一步阐述。

PROJECT 4: DEVELOPMENT OF NOVEL ANTIVIRALS TARGETING VIRAL RNA METHYLATION SUMMARY Coronaviruses cap their RNA by coordinated action of two methyltransferases (MTase): Nsp14, which catalyzes N7-guanidine methylation of GTP at the 5′ terminus of viral RNAs, and Nsp16, which forms C2′-O-methyl- ribosyladenine at the subsequent nucleotide. By mimicking mRNA of the host cell, the resulting cap structure is critical for immune evasion, stabilization of viral RNA and efficient translation. While loss of function mutations in the MTase domain of Nsp14 are lethal to SARS-CoV-2, SARS-CoV strains that carry mutant Nsp16 have low virulence, suggesting that targeting of these enzymes, either alone or in combination with other viral proteins, has strong therapeutic potential. In this application, we propose to develop antiviral agents that target MTase activities of Nsp14 and Nsp16. Our approach will assess the potential of RNA capping MTases as a novel target family for development of antiviral agents. Importantly, since both enzymes are conserved across coronaviruses known to infect humans, this approach could provide a footprint for development of pan-coronaviral acting agents. Inhibitors for each of the MTases will be identified using a combination of small molecule discovery approaches: computational docking, fragment linking and merging, and high throughput screening (HTS) to identify novel chemotypes. Enabled by the recent developments in availability of make-on-demand libraries, we propose to use ultra-large library docking to identify candidate inhibitors. Availability of drug-like compounds in in-house small molecule libraries will facilitate inhibitor identification through HTS. Hit compounds will be tested in a series of activity assays and validated using direct binding strategies. Experimentally determined structures of MTases in complex with small molecule inhibitors will be used to guide optimization, aided by access to make-on-demand libraries. The identified inhibitors will be prioritized based on their selectivity against a comprehensive panel of human MTases and antiviral activity in cellular models of SARS-CoV-2 infection. The subsequent medicinal chemistry optimization and assessment of antiviral activity in animal models is expected to result in Optimized Lead compounds, which will be further elaborated by our industry partners (Roche).