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：开发针对猪瘟病毒蛋白水解酶和聚合酶的抗病毒药物 冠状病毒、小核糖核酸病毒和本亚维尔病毒 摘要 该项目的目标是开发冠状病毒(CoV)、微小冠状病毒(PV)和布尼亚病毒(Bunyavirales)的类药物抑制剂 (BV)通过靶向病毒编码的基本蛋白酶(PR)和聚合酶(POL)进行复制。 正在研究的CoV的分子靶标是主要的PR(MPRO)、类木瓜蛋白酶的PR(PLP)和RNA- 依赖RNA聚合酶(RdRp)。PV的分子靶点是肠道病毒(EV)2A型和3C型PR和 RdRp，以及BV PLP和RdRp。在目标1和目标2中，我们将确定命中并进行领先优化，以 蛋白水解酶和RdRps。为了这两个目标，我们将探索其作用机制和抗性潜力。 尤其是针对新靶点的抑制剂。冠状病毒和冠状病毒之间的密切进化关系可能 还可以产生广谱抑制剂和两个病毒靶点之间的反馈。调查小组有一个 长期和成功的结构导向药物设计记录，包括广泛的PR和POL靶向。 我们已经建立了强大的、可扩展的表达系统来生产试剂量的SARS-CoV-2病毒 酵素。已开发出用于测量PR和RdRp活性的高通量、灵敏的分析方法 已用于发现共价和非共价的低μM Mpro的抑制剂，μM的PLP抑制剂，以及一种2A的生物激活剂。10万种化合物的生化 针对Lassa病毒RdRp的筛查已经产生了大量的点击和一条结构引导优化的途径。我们会 为相关的CoV、PV和BV开发强大的高通量(HTP)PR和RdRp分析。该衬底 PR的特异性将被分析以告知底物和抑制剂的设计，而细胞扰动则这些 将通过蛋白质组学和细胞断层扫描来探索抑制剂，以了解其作用机制。我们 将使用大量重组病毒和宿主PR和POL来快速评估HIT和铅的选择性 专一性。这些努力将得到八个技术核心的活动的支持。 努力将重点放在新的化学类型上，使用基于结构的对接、多样化和 多技术片段屏幕，以及HTS。将对化合物进行优化，以最大限度地减少最终的阻力。 将利用X射线建立结合模式和定量构效关系(QSAR) 结晶学、核磁共振光谱、低温电子显微镜和病毒复制分析。PR抑制剂和 RdRp抑制剂将一起进行测试，以确定相加或协同效应。这些信息将被用于 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发现的临床相关突变将被引入 野生型酶，并以其对我们新的化学类型的敏感性为特征。从这项工作中涌现出的 被一系列不同的抑制性化学类型和结构支架所促进的高效开发 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。