Transport Mechanisms and Inhibition of Efflux Pumps in Pathogenic Organisms

病原生物外排泵的转运机制和抑制

• 批准号: 10344321
• 负责人: SHOHEI KOIDE
• 金额: $ 74.35万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10344321
• 关键词: Active Biological Transport; Animals; Antibiotic Resistance; Antibiotics; Antibodies; Arginine; Aspartate; Binding; Biological Assay; Biological Models; Cells; Charge; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Cytoplasm; Data; Development; Directed Molecular Evolution; Drug Efflux; Drug Transport; Drug resistance; Effectiveness; Electrostatics; Fab Immunoglobulins; Free Energy; Glutamates; Goals; Growth; Human; Hybrids; Immunoglobulin Fragments; Infection; Ions; Knowledge; Methods; Microbiology; Molecular; Molecular Conformation; Mutation; NMR Spectroscopy; Organism; Pathogenicity; Peptides; Pharmaceutical Preparations; Phenotype; Play; Positioning Attribute; Protein Engineering; Proteins; Research; Resistance; Resolution; Role; Side; Specificity; Staphylococcus aureus; Structure; Synthesis Chemistry; System; Testing; Therapeutic; Toxic effect; Validation; antibiotic efflux; base; chemical synthesis; computational chemistry; design; drug resistant bacteria; efflux pump; experimental study; extracellular; global health; improved; inhibitor; insight; methicillin resistant Staphylococcus aureus; miniaturize; molecular recognition; multidrug transport; novel; pathogenic bacteria; peptidomimetics; protonation; resistance mechanism; structural biology; synthetic antibodies; tool; unnatural amino acids

Project Summary The long-term goal of our research is to develop first-in-class, protein-based inhibitors against human bacterial pathogens by directly blocking efflux pumps. Drug resistant bacteria pose an urgent global health challenge by reducing the effectiveness of antibiotics used to treat infections in humans and animals. The broadest resistance mechanism against antibiotics are efflux pumps, which transport drugs out of the cytoplasm and reduce toxicity to the organism. While it is known that efflux pumps display broad specificity to structurally distinct compounds, the mechanisms of polyspecific drug binding and ion-coupled transport remain unanswered questions in the field. Given the promiscuity of efflux pump binding to structurally distinct drugs, it is also unclear whether potent and selective efflux pump inhibitors can be designed to target specific classes of efflux pumps. The specific goals of this project are to discover novel mechanisms of active transport in drug resistant Staphylococcus aureus and to harness this knowledge to design selective inhibitors toward efflux pumps. Our proposal is strongly motivated by our recent discovery of antibody fragments (Fabs) that bind the Staphylococcus aureus efflux pump NorA and successful determination of high-resolution cryoEM structures using the Fabs as fiduciaries. The structures revealed that the Fabs insert a loop into the substrate binding pocket from the extracellular side, which suggests a design path toward protein- and peptide-based inhibitors. This interaction is facilitated by an electrostatic interaction between a positively charged arginine on the Fab and two essential anionic residues within NorA. Building on these preliminary data, we propose to carry out four Specific Aims. Aim 1 will develop a hybrid approach of cryo-electron microscopy and NMR spectroscopy to comprehensively study the transport cycle of NorA. Aim 2 will seek to determine the molecular basis for polyspecific drug binding. Aim 3 will design and characterize protein-based inhibitors that target the accessible, outward-open conformation of NorA. Aim 4 will develop peptides that miniaturize the antibody loops observed in the binding pocket of NorA. We have assembled an interdisciplinary team with expertise in structural biology, protein engineering, microbiology, chemical synthesis, and computational chemistry to rapidly answer fundamental questions about multidrug transport and inhibition of efflux pumps. All of the approaches applied to NorA will be translatable to other transporter systems.

项目摘要 我们研究的长期目标是开发一流的，基于蛋白质的抑制剂， 细菌病原体通过直接阻断外排泵。耐药细菌对全球健康构成紧迫威胁 通过降低用于治疗人类和动物感染的抗生素的有效性来应对挑战。的 最广泛的抗生素耐药机制是外排泵，它将药物转运出细胞质 并降低对生物体的毒性。虽然已知外排泵对结构上的 不同的化合物，多特异性药物结合和离子偶联转运的机制仍然没有答案 现场提问。考虑到外排泵与结构不同的药物结合的混杂性， 是否可以设计有效的和选择性的外排泵抑制剂以靶向特定类别的外排泵。 该项目的具体目标是发现耐药细胞主动转运的新机制 金黄色葡萄球菌，并利用这些知识来设计选择性抑制剂对外排泵。我们 我们最近发现的结合葡萄球菌的抗体片段（Fab）强烈地激发了这项提议 金黄色葡萄球菌外排泵诺拉和成功确定高分辨率cryoEM结构使用的Fab作为 受托人这些结构揭示了Fab将一个环插入底物结合口袋中， 细胞外侧，这表明了一个设计途径，以蛋白质和肽为基础的抑制剂。这种相互作用 通过Fab上的带正电荷的精氨酸和两个必需的精氨酸之间的静电相互作用来促进 诺拉内的阴离子残基。在这些初步数据的基础上，我们提出了四个具体目标。 目标1将开发一种低温电子显微镜和NMR光谱的混合方法， 研究诺拉的转运循环。目的2将寻求确定多特异性药物结合的分子基础。 目标3将设计和表征以蛋白质为基础的抑制剂，靶向可接近的，向外开放的构象 关于诺拉。目的4将开发使在诺拉的结合口袋中观察到的抗体环化的肽。 我们组建了一个跨学科的团队，他们拥有结构生物学，蛋白质工程， 微生物学、化学合成和计算化学，以快速回答以下基本问题 多药转运和外排泵抑制。应用于诺拉的所有方法都可以翻译为 其他运输系统。