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: 10615157
• 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/10615157
• 关键词: 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; 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; Retroviridae; Roentgen Rays; Role; SARS-CoV-2 inhibitor; SH2D3C gene; Specificity; Spectrum Analysis; Structure; System; Technology; United States National Institutes of Health; Viral; Virus; Virus Replication; X-Ray Crystallography; biophysical chemistry; cost effective; design; drug discovery; flexibility; human pathogen; influenza infection; influenzavirus; inhibitor therapy; 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.

项目摘要 该研究计划将调查理解构象多样性的一般假设 蛋白质的蛋白质将提供有关其生物学的新见解，并实现医学研究。它针对两个 系统类别：整体膜蛋白（IMP）和病毒宿主相互作用。小鬼扮演关键角色 守门员，接收器，转运蛋白，体内稳态调节剂和药物靶标。这些功能是介导的 通过在膜环境中imp的构象可塑性。小鬼是准备的挑战， 在适当的膜模仿环境中重新建立的挑战甚至更大。成本效益 凝结体积的同位素增强技术，将NMR与NMR结合的混合方法 进化共同变化（EC），新的接触预测方法和创新的建模方法 蛋白质结构预测界将应用于IMP的结构功能研究。这些小鬼， 从重要的人类病原体中进行选择，包括大肠杆菌，K。K。肺炎和铜绿假单胞菌 抗生素发现的靶标。 EC还将与NMR数据合并，以确定多重的结构 蛋白质的“本地状态”。我们程序的第二部分针对病毒 - 宿主生物分子 复合物和抗病毒药物发现。我们将利用创新的顺磁性NMR方法以及 小角度X射线散射（SAX），电子电子双共振光谱（鹿）和Förster 共振能量传递（FRET），严格定义动态域间结构分布 由鼠莫洛尼白血病病毒（MLV）整合酶（IN）的部分订购的接头。这些数据将是 在最大占用可能性（MAXOCC）的背景下进行解释，并用于探测此的作用 G-返回病毒基因整合机制的灵活性。域间接头也可以提供 约束伙伴滥交所需的灵活性。我们还将确定域间接头序列如何 影响Za非结构蛋白1（NS1）会议适当的可塑性，以定义其特异性和亲和力 宿主蛋白质和RNA。这种结构性和功能性滥交是NS1的机制的基础 抑制细胞先天免疫反应影响感染，并严格表征其 动态结构基础将为实时病毒疫苗开发提供基本信息。 我们还将应用我们的平台调查通过结合其主要蛋白的药物来抑制SARS-COV2病毒 （mpro）。我们已经确定了已经批准用于人类的三种药物，最初旨​​在抑制 NSP3/4A丙型肝炎病毒的蛋白酶，在低的病毒复制测定中也抑制SARS-COV2 微摩尔浓度。我们的计算对接研究还确定了其他几个FDA- 可能抑制MPRO的批准药物。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，以及 发挥其作为COVID-19疗法的潜力，或作为新疗法开发的铅化合物。