Structural Biology and Computational Modeling Core

结构生物学和计算建模核心

• 批准号: 10513917
• 负责人: LAWRENCE S SHAPIRO
• 金额: $ 532.66万
• 依托单位: HACKENSACK UNIVERSITY MEDICAL CENTER
• 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/10513917
• 关键词: 2019-nCoV; Affect; Alphavirus; Animal Model; Antiviral Agents; Architecture; Area; Binding; Biochemical; Biochemistry; Biophysics; COVID-19 treatment; Calorimetry; Caspase; Chemicals; Chikungunya virus; Collaborations; Complex; Computer Models; Cryoelectron Microscopy; Dengue; Development; Drug Kinetics; Drug Screening; Eastern Equine Encephalomyelitis; Epidemic; Epitopes; Excretory function; Exhibits; Exons; Exonuclease; Exoribonucleases; Family; Family member; Flavivirus; Genetic Transcription; Goals; Health; Hydrogen Bonding; In Vitro; Individual; Knowledge; Lead; Ligands; Literature; Macromolecular Complexes; Memorial Sloan-Kettering Cancer Center; Metabolism; Metagenomics; Methyltransferase; Middle East Respiratory Syndrome Coronavirus; Modification; Nonstructural Protein; Oral; Outcome; Outcome Study; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmacology; Pharmacotherapy; Preparation; Process; Property; Proteins; RNA; RNA Caps; RNA chemical synthesis; RNA purification; RNA-Directed RNA Polymerase; Recombinant Proteins; Research; Research Project Grants; Research Support; Roentgen Rays; SARS-CoV-2 inhibitor; SARS-CoV-2 protease; Services; Site-Directed Mutagenesis; Solubility; Structure; Structure-Activity Relationship; Surface; Surface Plasmon Resonance; System; Techniques; Technology; Thermodynamics; Toxic effect; Universities; Viral Genome; Virus; Virus Diseases; Virus Inhibitors; West Nile virus; Yellow Fever; ZIKA; Zika Virus; absorption; analog; base; biophysical properties; chikungunya; combinatorial; design; drug discovery; experience; improved; in silico; in vivo; inhibitor; innovation; lead optimization; member; metagenome; metropolitan; novel strategies; pandemic disease; process optimization; programs; protein complex; protein expression; protein protein interaction; protein purification; rational design; screening; small molecule; small molecule inhibitor; structural biology; viral RNA

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.

由Larry Shapiro和Dinshaw Patel领导的结构生物学（SB）核心将提供研究项目 具有生物化学、生物物理、结构和计算专业知识的团队，并为识别提供支持 和优化单独和组合靶向单亚基和多亚基的小分子抑制剂 SARS-CoV-2病毒基因组编码的非结构蛋白（Nsps）。这些目标包括半胱氨酸 蛋白酶3CLpro（Nsp 3）和PLpro（Nsp 5）和RNA依赖性RNA聚合酶（RdRp，Nsp 12-Nsp 7-Nsp 82） 对于病毒RNA转录和复制以及校正外切核酸酶（ExoN，Nsp 14）至关重要， 加帽甲基化酶N7-G-MT酶（Nsp 14-Nsp 10）和2 ′-O-MT酶（Nsp 16-Nsp 10），以及多亚基 复制-转录（RTC）复合物对于RNA加帽机制和合成保真度至关重要。 基于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。 主要集中在针对3CLpro蛋白酶的SARS-CoV-2抑制剂，项目6集中在抑制剂 而Patel实验室将支持项目3的研究，重点是SARS- CoV-2蛋白酶和甲基化酶抑制剂以及病毒RNA合成抑制剂项目4，包括RTC 复杂. SB Core预计将解决6至9个基于X射线的结构和3至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， 确定和化学修饰导致最有希望的候选物的优化。 目标1：SB Core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，然后进行详细的热力学（ITC和SPR）和结构（X射线和 cryo-EM）表征从实验和计算机模拟筛选中鉴定的小分子先导化合物， SARS-CoV-2的Nsps和靶向完整病毒的宏基因组筛选。 目标2：SB Core将分析靶标-靶标Nsp相互作用和构效关系， 促进基于计算的结构引导的先导化合物优化 测定和化学衍生化。这项工作涉及SB，Medicinal和 药理学核心，将针对表现出以下特征的优化电极导线的识别和表征： 针对一系列SARS-CoV-2 Nsp靶标的体外ADMET和体内药代动力学特性得到改善， 包括RTC综合体 目标3：SB核心将协助项目研究工作，以扩展正在进行的针对SARS的抑制剂研究- CoV-2 Nsps与黄病毒和甲病毒家族的类似成员。结构派生的比较 病毒家族中的通道蛋白复合物的口袋结构和分子间接触应该 确定靶向SARS-CoV-2的Nsps的抑制剂在多大程度上需要额外的修饰以用于靶向 它们在黄病毒和甲病毒家族成员中的类似Nsp。