Developing Antivirals Targeting Proteases and Polymerases of Coronaviruses, Picornaviruses and Bunyavirales

项目2：靶向蛋白酶和聚合酶的抗病毒药 冠状病毒、小冠状病毒和布尼亚病毒 总结 本项目的目标是开发冠状病毒（CoV）、小核糖核酸病毒（PV）和布尼亚病毒目的药物样抑制剂 (BV)通过靶向病毒编码的必需蛋白酶（PR）和聚合酶（Pol）来复制。 所追求的CoV的分子靶标是主要PR（MPro）、木瓜蛋白酶样PR（PLP）和RNA-聚合酶。 依赖性RNA聚合酶（RdRp）。PV的分子靶点是肠道病毒（EV）2A和3C PR， RdRp，以及BV PLP和RdRp。在目标1和目标2中，我们将识别命中并进行潜在客户优化， 蛋白酶和RdRps。将针对这两个目标探索作用机制和抗性潜力， 特别是对于靶向新位点的抑制剂。冠状病毒和肺静脉之间的密切进化关系可能 也产生广谱抑制剂和两种病毒靶标之间的反馈。调查小组有一个 在结构导向药物设计方面有着长期而成功的记录，包括PR和Pol的广泛靶向。 我们已经建立了强大的，可扩展的表达系统，用于生产试剂量的SARS-CoV-2病毒 内切酶已经开发了用于测量PR和RdRp活性的高通量、灵敏的测定， SARS-CoV-2 MPro、PLP和EV 2A，并已用于发现共价和非共价低μM MPro抑制剂、PLP μM抑制剂和2A生物激活剂。一种十万种化合物的生化制剂 针对拉沙病毒RdRp的筛选已经产生了大量的命中和结构导向优化的路径。我们将 为相关CoV、PV和BV开发稳健的高通量（HTP）PR和RdRp检测。基板 PR的特异性将被分析以告知底物和抑制剂设计，而细胞扰动这些 将通过蛋白质组学和细胞断层扫描来探索抑制剂赋予以了解作用机制。我们 将使用大量重组病毒和宿主PR和Pol来快速评估命中和先导选择性， 的特异性这些努力将得到八个技术核心活动的支持。 努力将集中在新的化学型，确定使用基于结构的对接，多样性和 多技术碎片筛选和HTS。化合物将被优化，以最大限度地减少最终的阻力。 结合模式和定量构效关系（QSAR）将使用X射线 晶体学、NMR光谱学、低温电子显微镜和病毒复制测定。PR抑制剂和 将一起测试RdRp抑制剂以确定累加或协同效应。此信息将用于 指导下一轮筛选和抑制剂改进。 在接受PR或RdRp抑制剂治疗的患者中鉴定出的临床相关突变将被引入到 野生型酶，并表征其对我们的新化学型的敏感性。通过这项工作， 是一个多样化的阵列抑制化学型和结构支架，以促进发展的高效 毒品虽然雄心勃勃，但广泛的初步成功支持了这些目标的实用主义。

PROJECT 2: DEVELOPING ANTIVIRALS TARGETING PROTEASES AND POLYMERASES OF CORONAVIRUSES, PICORNAVIRUSES AND BUNYAVIRALES SUMMARY The goal of this Project is to develop drug-like inhibitors of coronavirus (CoV), picornavirus (PV) and Bunyavirales (BV) replication by targeting essential protease (PR) and polymerase (Pol) enzymes encoded by the viruses. Molecular targets of the CoVs being pursued are the main PR (MPro), the papain-like PR (PLP), and the RNA- dependent RNA polymerase (RdRp). Molecular targets of the PVs are the enteroviral (EV) 2A and 3C PRs and RdRp, and for BV PLP and RdRp. In Aims 1 and 2 we will identify hits and conduct lead optimization, for proteases and RdRps, respectively. Mechanism of action and resistance potential will be explored for both aims, especially for inhibitors that target novel sites. The close evolutionary relationship between CoVs and PVs may also yield broad-spectrum inhibitors and feedback between both viral targets. The team of investigators have a long and successful track record of structure-guided drug design, including extensive targeting of PRs and Pols. We have established robust, scalable expression systems for producing reagent quantities of SARS-CoV-2 viral enzymes. High-throughput, sensitive assays for measuring PR and RdRp activity have been developed for SARS-CoV-2 MPro, PLP and EV 2A and have been used to discover both covalent and noncovalent low μM inhibitors for MPro, μM inhibitors for PLP, and a biologic activator of 2A. A 100,000-compound biochemical screen against Lassa virus RdRp has yielded numerous hits and a path to structure-guided optimization. We will develop robust high-throughput (HTP) PR and RdRp assays for related CoVs, PVs, and BVs. The substrate specificity of PRs will be profiled to inform substrate and inhibitor design, while cellular perturbations these inhibitors confer will be explored by proteomics and cellular tomography to understand mechanism of action. We will use large panels of recombinant viral and host PRs and Pols to rapidly evaluate hit and lead selectivity and specificity. These efforts will be supported by the activities of the eight Technology Cores. Efforts will focus on novel chemotypes, identified using a combination of structure-based docking, diverse and multi-technique fragment screens, and HTS. Compounds will be optimized to minimize eventual resistance. Mode of binding and quantitative structure-activity relationships (QSAR) will be established using X-ray crystallography, NMR spectroscopy, cryo-electron microscopy and viral replication assays. PR inhibitors and RdRp inhibitors will be tested together to identify additive or synergistic effects. This information will be used to direct the next round of screening and inhibitor improvement. Clinically relevant mutations identified in patients treated with PR or RdRp inhibitors will be introduced into the wild-type enzymes and characterized for their sensitivity to our novel chemotypes. Emerging from this work will be a diverse array of inhibitory chemotypes and structural scaffolds to facilitate development of highly effective drugs. While ambitious, extensive preliminary success supports the pragmatism of these aims.