Structural Biology and Computational Modeling Core

由Larry Shapiro和Dinshaw Patel领导的结构生物学(SB)核心将提供研究项目 具有生化、生物物理、结构和计算专业知识并支持鉴定的团队 以及单独和组合靶向单亚基和多亚基的小分子抑制剂的优化 SARS-CoV-2病毒基因组编码的非结构蛋白(NSP)。这些目标包括半胱氨酸 蛋白水解酶3CLPro(NSP3)和PLPro(Nsp5)和依赖RNA的RNA聚合酶(RdRp，Nsp12-Nsp7-Nsp82) 对病毒RNA转录和复制以及校对外切酶(外显子，Nsp14)至关重要， 封端甲基酶N7-G-MTase(Nsp14-Nsp10)和2‘-O-MTase(Nsp16-Nsp10)，以及多亚基 复制-转录(RTC)复合体对RNA封帽机制和合成保真度至关重要。 根据正在进行的和预期的合作，夏皮罗实验室将支持项目1、2和5的研究 主要关注针对3CLpro蛋白酶的SARS-CoV-2抑制剂和项目6专注于抑制剂 而Patel实验室将支持以SARS为重点的项目3的研究- CoV-2蛋白酶和甲基酶抑制剂和包括RTC在内的病毒RNA合成抑制剂的项目4 很复杂。SB Core预计将解决6到9个基于X射线的结构和3到5个基于冷冻-EM的结构 作为这项工作的一部分，同时还提供指导结构迭代周期的计算专业知识 测定和化学修饰导致最有希望的候选者的优化。 目标1：SB核心将协助项目在蛋白质表达和纯化以及RNA方面的研究工作 转录和纯化，随后是详细的热力学(ITC和SPR)和结构(X-射线和 低温电子显微镜)表征从实验和硅屏中识别的小分子铅 SARS-CoV-2的NSP和针对完整病毒的元基因组筛选。 目标2：SB核心将分析抑制剂-靶NSP相互作用和结构-活性(SAR)关系，以 简化涉及结构迭代循环的基于计算的结构引导先导化合物优化 测定和化学衍生化。这一努力涉及SB、Medical和 药理学核心，将以识别和表征展示的优化线索为目标 改善了针对一系列SARS-CoV-2 NSP靶点的体外和体内药代动力学特性， 包括RTC建筑群。 目标3：SB核心将协助项目研究工作，以扩大正在进行的针对SARS的抑制剂的研究- CoV-2 NSPS与黄病毒和甲病毒家族的类似成员。结构派生法的比较研究 病毒家族间抑制蛋白复合体的口袋结构和分子间接触 确定靶向SARS-CoV-2 NSP的抑制剂需要在多大程度上进行额外的靶向修饰 它们在黄病毒和甲病毒家族成员中的相似NSP。

The Structural Biology (SB) Core headed by Larry Shapiro and Dinshaw Patel will provide Research Project teams with biochemical, biophysical, structural and computational expertise and support towards identification and optimization of small-molecule inhibitors that individually and combinatorially target single- and multisubunit nonstructural proteins (Nsps) encoded by the SARS-CoV-2 viral genome. These targets include cysteine proteases 3CLpro (Nsp3) and PLpro (Nsp5) and RNA-dependent RNA polymerase (RdRp, Nsp12-Nsp7-Nsp82) critical for viral RNA transcription and replication, as well as the proofreading exonuclease (ExoN, Nsp14), the capping methylases N7-G-MTase (Nsp14-Nsp10) and 2’-O-MTase (Nsp16-Nsp10), and the multisubunit replication-transcription (RTC) complex critical for RNA capping machinery and synthesis fidelity. Based on ongoing and anticipated collaborations, the Shapiro lab will support research on projects 1, 2 and 5 focused primarily on SARS-CoV-2 inhibitors targeting 3CLpro protease and project 6 focused on inhibitors identified from metagenome screens, while the Patel lab will support research on project 3 focused on SARS- CoV-2 protease and methylase inhibitors and project 4 on inhibitors of viral RNA synthesis, including the RTC complex. The SB Core anticipates solving 6 to 9 X-ray based structures and 3 to 5 cryo-EM-based structures per year as part of this effort, while also providing computational expertise guiding iterative cycles of structure determination and chemical modification leading to optimization of the most promising candidates. Aim 1: The SB Core will assist Project research efforts in protein expression and purification, as well as RNA transcription and purification, followed by a detailed thermodynamic (ITC and SPR) and structural (X-ray and cryo-EM) characterization of small molecule leads identified from experimental and in silico screens targeted to Nsps of SARS-CoV-2 and metagenomic screens targeted to the intact virus. Aim 2: The SB Core will analyze inhibitor-target Nsp interactions and structure-activity (SAR) relationships to facilitate computation-based structure-guided lead compound optimization involving iterative cycles of structure determination and chemical derivatization. This effort, involving close collaboration between SB, Medicinal and Pharmacology Cores, will be targeted to identification and characterization of optimized leads that exhibit improved in vitro ADMET and in vivo pharmacokinetic properties against a range of SARS-CoV-2 Nsp targets, including the RTC complex. Aim 3: The SB Core will assist Project research efforts to extend ongoing studies on inhibitors targeting SARS- CoV-2 Nsps to analogous members of the flavivirus and alphavirus families. A comparison of structure-derived pocket architectures and intermolecular contacts for inhibitor-protein complexes amongst virus families should determine to what extent inhibitors targeted to Nsps of SARS-CoV-2 need additional modifications for targeting their analogous Nsps in flavivirus and alphavirus family members.