Development of Novel Antivirals Targeting Viral RNA Methylation

项目4：开发靶向病毒RNA甲基化的新型抗病毒药物 总结 冠状病毒通过两种甲基转移酶（MTase）的协调作用来覆盖其RNA：Nsp 14，其催化 病毒RNA 5 '末端的GTP和Nsp 16的N7-胍甲基化，形成C2'-O-甲基- 核糖基腺嘌呤在随后的核苷酸。通过模拟宿主细胞的mRNA， 对于免疫逃避、病毒RNA的稳定和有效翻译至关重要。虽然功能丧失突变 Nsp 14的MTase结构域中的突变体对SARS-CoV-2具有致死性，携带突变Nsp 16的SARS-CoV株具有低的 毒力，这表明这些酶的靶向，无论是单独或与其他病毒蛋白质， 具有很强的治疗潜力。在本申请中，我们建议开发靶向MTase的抗病毒剂 NSP 14和NSP 16的活性。我们的方法将评估RNA加帽MTases作为新靶点的潜力 开发抗病毒药物的家族。重要的是，由于这两种酶在冠状病毒中是保守的， 已知感染人类，这种方法可以为泛冠状病毒作用的发展提供足迹 剂. 将使用小分子发现方法的组合鉴定每种MT酶的抑制剂： 计算对接，片段连接和合并，以及高通量筛选（HTS），以确定新的 化学型随着随需应变图书馆的发展，我们建议 使用超大库对接来鉴定候选抑制剂。内部药物样化合物的可用性 小分子文库将有助于通过HTS鉴定抑制剂。命中化合物将在一系列测试 的活性测定和验证使用直接结合策略。实验确定的MT酶结构 在与小分子抑制剂的复合物中，将用于指导优化，并通过按需制造来辅助 图书馆.确定的抑制剂将根据其对一组全面的 SARS-CoV-2感染细胞模型中的人类MT酶和抗病毒活性。随后的药物 在动物模型中进行化学优化和抗病毒活性评估，预计将导致优化 先导化合物，将由我们的行业合作伙伴（罗氏）进一步开发。

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).