Biochemistry core

核心1：生物化学 摘要 为了支持QCRG大流行应对计划的总体和项目目标，生化核心将 开发、优化和执行病毒蛋白的表达、纯化和活性分析，用于结构和 功能特性。这包括以以下格式适当地呈现病毒蛋白：(1)高分辨率 单个蛋白质和多亚基复合体的结构测定；(2)筛选有效的Hit和Lead 化合物；以及(3)结构基铅的HIT和铅化合物共结构测定 优化和药物发现。QCRG大流行应对计划项目组合目前专注于 关于具有大流行潜力的病毒的蛋白质和具有高药用潜力的蛋白质。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)，托加病毒(基孔肯雅病毒，辛德比斯，塞姆利基森林，罗斯河)，以及 微小核糖核酸病毒(EV-D68、EV-A71)。具体地说，我们将提供一致的、高水平的蛋白质纯化 冠状病毒(如NSP3、NSP5、NSP7、NSP8、NSP12)的蛋白酶(PR)和聚合酶(POL)靶标 肠道病毒(例如，2A和3Dpol1)(项目2)；E和M冠状病毒膜蛋白，以及病毒素‘6K’ 来自Togaviridae Chikungenya、Sindbis、Semliki森林、罗斯河的甲型病毒的蛋白质(项目3)； 冠状病毒甲基转移酶(例如Nsp10、Nsp14和Nsp16)(项目4)；来自 SARS-CoV-2和基孔肯雅病(项目5)；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)。我们已经表达了SARS-CoV-2编码的23个蛋白中的15个 建立了这些和开发的技术的可行性，这些技术可以适应和发展到表达 以及从其他病毒中提纯所有预期目标(摘要见研究战略，表1)。

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).