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

开发针对冠状病毒、小核糖核酸病毒和布尼亚病毒的蛋白酶和聚合酶的抗病毒药物

• 批准号: 10512628
• 负责人: Charles Scott Craik
• 金额: $ 516.83万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512628
• 关键词: 2019-nCoV; Acquired Immunodeficiency Syndrome; Affinity; Animal Model; Antiviral Agents; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Bunyavirales; COVID-19 treatment; Caspase; Cell Culture Techniques; Cell model; Cells; Chemicals; Collection; Combined Modality Therapy; Communicable Diseases; Complex; Congo; Coronavirus; Cryoelectron Microscopy; Crystallization; Crystallography; Development; Docking; Drug Design; Drug Targeting; Drug resistance; Enzyme Inhibition; Enzymes; Family; Family Picornaviridae; Feedback; Genetic Transcription; Genome; Goals; HIV; HIV Protease Inhibitors; Head; Immune system; In Vitro; Infection; Influenza; Lassa virus; Lead; Letters; Libraries; Measures; Modeling; Molecular Target; Mutation; NMR Spectroscopy; Neuraminidase inhibitor; Nonstructural Protein; Oral; Papain; Patients; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmaceutical Preparations; Polymerase; Protease Inhibitor; Proteomics; Quantitative Structure-Activity Relationship; RNA Polymerase Inhibitor; RNA Viruses; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Reagent; Recombinants; Research Personnel; Resistance; Role; Sin Nombre virus; Site; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; System; Techniques; Technology; Testing; Ubiquitination; Variant; Viral; Virus; Virus Replication; Work; X-Ray Crystallography; base; candidate selection; chemotherapy; clinically relevant; design; drug development; drug discovery; experience; experimental study; hemorrhagic fever virus; in vitro Assay; in vivo; inhibitor; lead optimization; member; next generation; novel; pandemic disease; polypeptide; preclinical development; programs; response; scaffold; screening; structural biology; success; synergism; tomography; viral RNA; viral resistance; virology

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.

项目2：开发针对蛋白酶和聚合酶的抗病毒药 冠状病毒，Picornaviruses和Bunyavirales 概括 该项目的目的是开发冠状病毒（COV），Picornavirus（PV）和Bunyavirales的药物状抑制剂 （BV）通过靶向由病毒编码的必需蛋白酶（PR）和聚合酶（POL）酶复制。 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- 依赖性RNA聚合酶（RDRP）。 PV的分子靶标是肠病毒（EV）2A和3C PR和 RDRP，以及BV PLP和RDRP。在目标1和2中，我们将确定打击并进行铅优化，因为 蛋白酶和RDRP。两个目标都将探索动作机理和抵抗潜力的机理 特别是针对针对新部位的抑制剂。 COV和PV之间的密切进化关系可能 还会产生两个病毒靶标之间的广谱抑制剂和反馈。调查人员团队有一个 结构引导的药物设计的长期成功记录，包括广泛的PR和POL靶向。 我们已经建立了可实现的可扩展表达系统，用于生产大量SARS-COV-2病毒 酶。已经开发了用于测量PR和RDRP活动的高通量，敏感测定 SARS-COV-2 MPRO，PLP和EV 2A，已用于发现共价和非共价低μm MPRO的抑制剂，PLP的μM抑制剂和2A的生物激活剂。 100,000个化合物的生化 针对LASSA病毒RDRP的筛查产生了许多命中和结构引导优化的途径。我们将 为相关的COV，PVS和BVS开发强大的高通量（HTP）PR和RDRP分析。基板 PR的特异性将被介绍以告知底物和抑制剂设计，而细胞扰动这些 蛋白质组学和细胞断层扫描将探索抑制剂会议，以了解作用机理。我们 将使用大型重组病毒和宿主PR和POL的大型面板来快速评估命中和铅选择性，并且 特异性。这些努力将得到八个技术核心的活动的支持。 努力将专注于新型化学型，使用基于结构的对接，多样和 多技术片段屏幕和HTS。化合物将被优化以最大程度地减少事件电阻。 将使用X射线建立结合和定量结构活性关系（QSAR）的模式 晶体学，NMR光谱，冷冻电子显微镜和病毒复制测定法。 PR抑制剂和 RDRP抑制剂将共同测试以识别添加剂或协同作用。此信息将用于 指导下一轮筛查和抑制剂改进。 在接受PR或RDRP抑制剂治疗的患者中发现的临床相关突变将引入 野生型酶的特征是它们对我们新型化学型的敏感性。从这项工作中出现 成为抑制性化学型和结构支架的潜水员阵列，以促进高效的发展 毒品。尽管雄心勃勃，广泛的初步成功支持了这些目标的实用主义。