Biochemistry core

生化核心

• 批准号: 10512619
• 负责人: Robert M Stroud
• 金额: $ 158.11万
• 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/10512619
• 关键词: 2019-nCoV; Address; Alphavirus; Biochemical; Biochemistry; Biological Assay; Cells; Chikungunya virus; Code; Complex; Coronavirus; Coronavirus nucleocapsid protein; Cryo-electron tomography; Crystallography; Dengue; Drug Targeting; Electron Microscopy; Elements; Enterovirus; Enterovirus 68; Enzymes; Escherichia coli; Family; Family Picornaviridae; Flavivirus; Genome; Goals; Grant; HIV; Human; Human Parainfluenza Virus 4; Immune response; Individual; Influenza; Insecta; Integral Membrane Protein; Label; Lead; Mammalian Cell; Membrane; Membrane Proteins; Methyltransferase; Middle East Respiratory Syndrome Coronavirus; Natural Immunity; Nuclear Magnetic Resonance; Nucleocapsid; Nucleocapsid Proteins; Paramyxovirus; Pathway interactions; Peptide Hydrolases; Polymerase; Process; Production; Proteins; Protocols documentation; RNA; Research; Resolution; Rivers; Ross river virus; S-Adenosylmethionine; SARS coronavirus; Sampling; Semliki forest virus; Sindbis Virus; Structural Protein; Structure; System; Technology; Togaviridae; Viral; Viral Matrix Proteins; Viral Proteins; Virion; Virus; Virus Diseases; Virus Inactivation; ZIKA; Zika Virus; assay development; base; chikungunya; cofactor; cryogenics; drug discovery; experience; forest; high throughput screening; human coronavirus; inhibitor; lead optimization; multiple myeloma M Protein; pandemic disease; parainfluenza virus; prevent; programs; protein E; protein expression; protein purification; protein structure; response; screening; small molecule

CORE 1: BIOCHEMISTRY SUMMARY In support of QCRG Pandemic Response Program overall and Project goals, the Biochemistry Core will develop, optimize and execute viral protein expression, purification, and activity assays for structure and functional characterization. This includes suitable presentation of viral proteins in formats for: (1) high resolution structure determination of individual proteins and multi-subunit complexes; (2) screening effective hit and lead compounds; and (3) co-structure determination with hit and lead compounds for structure-based lead optimization and drug discovery. The QCRG Pandemic Response Program Project portfolio currently focuses on proteins from viruses with pandemic potential and proteins that have high potential for druggability. These viruses include 7 species of human coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, OC43, NL63, HKU1, 229E), flaviviruses (dengue, zika), togaviruses (chikungunya virus, Sindbis, Semliki forest, Ross river), and picornaviruses (EV-D68, EV-A71). Specifically, we will provide consistent, high level protein purification of protease (PR) and polymerase (Pol) targets from coronaviruses (e.g. Nsp3, Nsp5, Nsp7, Nsp8, Nsp12) and enteroviruses (e.g. 2A and 3Dpol) (Project 2); E and M coronavirus membrane proteins, and the viroporin '6K' protein from Alphaviruses of the Togaviridae chikungunya, Sindbis, Semliki forest, Ross river (Project 3); coronavirus methyltransferases (e.g. Nsp10, Nsp14, and Nsp16) (Project 4); the macrodomain of Nsp3 from SARS-CoV-2 and chikungunya (Project 5); and N and Orf9b from coronaviruses, and the N protein of zika, dengue, and HPIV3 (Project 6). Having expressed 15 proteins from the 23 coded by SARS-CoV-2 we have established feasibility for these and developed technologies that can be adapted and evolved toward expression and purification for all intended targets from other viruses (for summary see Research Strategy, Table 1).

核心1：生物化学 概括 为了支持 QCRG 流行病应对计划的总体目标和项目目标，生物化学核心将 开发、优化和执行病毒蛋白表达、纯化以及结构和活性测定 功能表征。这包括以以下格式适当呈现病毒蛋白：(1) 高分辨率 单个蛋白质和多亚基复合物的结构测定； (2)筛选有效命中和领先 化合物； (3) 用命中化合物和先导化合物确定基于结构的先导化合物的共结构 优化和药物发现。 QCRG 流行病应对计划项目组合目前重点关注 研究来自具有大流行潜力的病毒的蛋白质和具有高成药潜力的蛋白质。这些 病毒包括7种人类冠状病毒（SARS-CoV-2、SARS-CoV、MERS-CoV、OC43、NL63、HKU1、 229E）、黄病毒（登革热、寨卡病毒）、披膜病毒（基孔肯雅病毒、辛德比斯病毒、塞姆利基森林病毒、罗斯河病毒）和 小核糖核酸病毒（EV-D68、EV-A71）。具体来说，我们将提供一致的、高水平的蛋白质纯化 冠状病毒的蛋白酶 (PR) 和聚合酶 (Pol) 靶标（例如 Nsp3、Nsp5、Nsp7、Nsp8、Nsp12）和 肠道病毒（例如 2A 和 3Dpol）（项目 2）； E 和 M 冠状病毒膜蛋白以及病毒孔蛋白“6K” 来自基孔肯雅披膜病毒科、辛德比斯、塞姆利基森林、罗斯河的甲病毒的蛋白质（项目 3）； 冠状病毒甲基转移酶（例如 Nsp10、Nsp14 和 Nsp16）（项目 4）； Nsp3 的宏结构域 SARS-CoV-2 和基孔肯雅热（项目 5）；以及来自冠状病毒的 N 和 Orf9b，以及寨卡病毒的 N 蛋白， 登革热和 HPIV3（项目 6）。我们表达了 SARS-CoV-2 编码的 23 种蛋白质中的 15 种 确定了这些技术的可行性，并开发了可以适应和发展到表达的技术 以及从其他病毒中纯化所有预期目标（摘要参见研究策略，表 1）。