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 总结 为了支持QCRG大流行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、纯化和活性测定， 功能特性这包括病毒蛋白质的适当呈现，其形式为：（1）高分辨率 单个蛋白质和多亚基复合物的结构测定：（2）筛选有效的命中和引导 化合物;和（3）与命中和铅化合物的共结构测定，用于基于结构的铅 优化和药物发现。QCRG流行病应对计划项目组合目前侧重于 对来自具有大流行潜力的病毒的蛋白质和具有高药物潜力的蛋白质进行研究。这些 病毒包括7种人类冠状病毒（SARS-CoV-2，SARS-CoV，MERS-CoV，OC 43，NL 63，HKU 1， 229E）、黄病毒（登革热、寨卡病毒）、披膜病毒（基孔肯雅病毒、辛德毕斯病毒、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， 来自冠状病毒的蛋白酶（PR）和聚合酶（Pol）靶标（例如Nsp 3、Nsp 5、Nsp 7、Nsp 8、Nsp 12），以及 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、辛德毕斯、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Nsp 10、Nsp 14和Nsp 16）（项目4）;来自 SARS-CoV-2和基孔肯雅热（项目5）;冠状病毒的N和Orf9b，以及寨卡病毒的N蛋白， 登革热和HPIV 3（项目6）。在表达了SARS-CoV-2编码的23个蛋白中的15个蛋白后， 为这些和开发的技术建立可行性，这些技术可以适应和发展， 并从其他病毒中纯化所有预期靶标（总结见研究策略，表1）。