Hybrid Methods for Dynamic Structure Analysis of Proteins from Pathogenic Microorganisms

病原微生物蛋白质动态结构分析的混合方法

• 批准号: 10205615
• 负责人: GAETANO T MONTELIONE
• 金额: $ 65.8万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205615
• 关键词: 2019-nCoV; Affinity; Antibiotics; Antiviral Agents; Attenuated Live Virus Vaccine; Binding; Biological Assay; Biology; COVID-19 therapeutics; Communities; Complex; Coronavirus; Data; Docking; Drug Targeting; Electrons; Environment; Enzyme Kinetics; Escherichia coli; FDA approved; Fluorescence Resonance Energy Transfer; Gammaretrovirus; Genes; Genome; Hepatitis C virus; Homeostasis; Human; Hybrids; Influenza; Innate Immune Response; Integral Membrane Protein; Integrase; Isotopes; Klebsiella pneumoniae; Lead; Mediating; Medical Research; Membrane; Methods; Modeling; Molecular Conformation; Moloney Leukemia Virus; Nonstructural Protein; Pathogenicity; Peptide Hydrolases; Pharmaceutical Preparations; Play; Probability; Protease Inhibitor; Protein Analysis; Proteins; Pseudomonas aeruginosa; RNA; Reproducibility; Research Project Summaries; Roentgen Rays; Role; SH2D3C gene; Specificity; Spectrum Analysis; Structure; System; Technology; United States National Institutes of Health; Variant; Viral; Virus; Virus Replication; X-Ray Crystallography; biophysical chemistry; cost effective; design; drug discovery; flexibility; human pathogen; influenza infection; influenzavirus; innovation; insight; microorganism; novel; novel therapeutics; pathogenic bacteria; priority pathogen; programs; protein structure prediction; receptor; reconstitution; therapeutic development; vaccine development

PROJECT SUMMARY This research program will investigate the general hypothesis that understanding the conformational diversity of proteins will provide new insights into their biology, and enable medical research. It is directed to two classes of systems: Integral Membrane Proteins (IMPs) and viral-host interactions. IMPs play critical roles as gate keepers, receptors, transporters, homeostasis regulators, and drug targets. These functions are mediated by the conformational plasticity of the IMP in the membrane environment. IMPs are challenging to prepare, and even more challenging to reconstitute in appropriate membrane mimicking environments. Cost-effective technologies for isotope-enrichment in condensed volumes, hybrid approaches combining NMR with evolutionary co-variation (ECs), novel methods of contact prediction, and innovative modeling methods from the protein structure prediction community, will be applied to structure-function studies of IMPs. These IMPs, chosen from important human pathogens, including E. coli, K. pneumoniae, and P. aeruginosa, are potential targets for antibiotic discovery. ECs will also be combined with NMR data to determine structures of multiple “native states” of proteins. The second component of our program is directed to viral – host biomolecular complexes, and antiviral drug discovery. We will utilize innovative paramagnetic NMR methods, together with small angle X-ray scattering (SAXS), electron-electron double resonance spectroscopy (DEER), and Förster resonance energy transfer (FRET), to rigorously define dynamic interdomain structural distributions conferred by the partially-ordered linkers of the murine Moloney Leukemia Virus (MLV) integrase (IN). These data will be interpreted in the context of maximum occupancy probabilities (MaxOcc), and used to probe the role(s) of this flexibility in the gene integration mechanisms of g-retroviruses. Interdomain linkers also function to provide flexibility needed for binding partner promiscuity. We will also determine how the interdomain linker sequences of influenza Non-Structural Protein 1 (NS1) confer appropriate plasticity to define its specificity and affinity for host proteins and RNAs. This structural and functional promiscuity underlies NS1’s mechanisms for suppressing the cellular innate immune response to influenza infection, and rigorous characterization of its dynamic structural basis will provide fundamental information for live-attenuated virus vaccine development. We will also apply our platform to investigate drugs that inhibit SARS-CoV2 virus by binding its main protease (Mpro). We have identified three drugs, already approved for use in humans, originally designed to inhibit the NSP3/4A protease of hepatitis C virus, that also inhibit SARS-CoV2 in viral replication assays at low micromolar concentrations. Our computational docking studies have also identified several other FDA- approved drugs that may inhibit Mpro. Enzyme kinetic, biophysical chemistry, and X-ray crystallography studies will be used to characterize complexes formed between these protease inhibitor drugs and Mpro, and to develop their potential as COVID-19 therapeutics, or as lead compounds for new therapeutic development.

项目概要 该研究计划将调查了解构象多样性的一般假设 蛋白质的研究将为其生物学提供新的见解，并促进医学研究。它针对两个方面。 系统类别：整合膜蛋白 (IMP) 和病毒-宿主相互作用发挥着关键作用。 守门者、受体、转运蛋白、稳态调节剂和药物靶点这些功能是介导的。 由于 IMP 在膜环境中的构象可塑性，其制备具有挑战性，并且 在适当的膜模拟环境中重建更具挑战性。 浓缩体积同位素富集技术，将 NMR 与 进化共变（EC）、接触预测的新方法以及创新的建模方法 蛋白质结构预测社区将应用于 IMP 的结构功能研究。 选自重要的人类病原体，包括大肠杆菌、肺炎克雷伯菌和铜绿假单胞菌，是潜在的 抗生素发现的目标也将与 NMR 数据相结合，以确定多种结构。 我们计划的第二个组成部分针对病毒——宿主生物分子。 我们将利用创新的顺磁核磁共振方法以及抗病毒药物的发现。 小角 X 射线散射 (SAXS)、电子-电子双共振光谱 (DEER) 和 Förster 共振能量转移（FRET），严格定义赋予的动态域间结构分布 通过鼠莫洛尼白血病病毒 (MLV) 整合酶 (IN) 的部分有序连接体这些数据将是。 在最大占用概率 (MaxOcc) 的背景下进行解释，并用于探测此的角色 g-逆转录病毒的基因整合机制的灵活性也起到提供作用。 我们还将确定域间接头的序列方式。 流感非结构蛋白 1 (NS1) 赋予适当的可塑性，以确定其特异性和亲和力 这种结构和功能的混杂是 NS1 机制的基础。 抑制细胞对流感感染的先天免疫反应，及其严格的表征 动态结构基础将为减毒活病毒疫苗的开发提供基础信息。 我们还将应用我们的平台来研究通过结合其主要蛋白酶来抑制 SARS-CoV2 病毒的药物 （Mpro）。我们已经确定了三种药物，已批准用于人类，最初设计用于抑制 丙型肝炎病毒的 NSP3/4A 蛋白酶，在低病毒检测复制中也能抑制 SARS-CoV2 我们的计算对接研究还确定了其他几种 FDA- 批准的药物可能抑制 Mpro 酶动力学、生物物理化学和 X 射线晶体学研究。 将用于表征这些蛋白酶抑制剂药物和 Mpro 之间形成的复合物，并 开发它们作为 COVID-19 疗法的潜力，或作为新疗法开发的先导化合物。