Transport Mechanisms and Inhibition of Efflux Pumps in Pathogenic Organisms

项目摘要 我们研究的长期目标是开发一流的、以蛋白质为基础的针对人类的抑制剂 通过直接阻断外排泵来抑制细菌病原体。抗药性细菌构成全球紧迫的健康问题 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。这个 对抗生素最广泛的耐药机制是外排泵，它将药物输送到细胞质外。 并减少对生物体的毒性。虽然已知外排泵在结构上表现出广泛的特异性 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 在现场的问题。考虑到外排泵与结构不同的药物结合的混杂，它也不清楚。 强效和选择性外排泵抑制剂是否可以针对特定类别的外排泵而设计。 这个项目的具体目标是发现抗药性中主动转运的新机制。 金黄色葡萄球菌，并利用这一知识来设计选择性的外排泵抑制剂。我们的 我们最近发现的与葡萄球菌结合的抗体片段(Fabs)强烈地推动了这一提议 Areus外排泵NORA和使用Fabs AS成功确定高分辨率低温电子显微镜结构 受托人。结构显示，纤维从底物结合袋中插入一个环 胞外方面，这表明了一条以蛋白质和多肽为基础的抑制剂的设计路径。这种互动 是由FAB上带正电荷的精氨酸和两种必需的 Nora内的阴离子残留物。在这些初步数据的基础上，我们建议实现四个具体目标。 目标1将发展冷冻电子显微镜和核磁共振光谱的混合方法，以全面 研究诺拉的运输周期。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，目标是可访问的、向外开放的构象 关于诺拉的。目标4将开发多肽，使在NorA结合口袋中观察到的抗体环变得小型化。 我们已经组建了一支跨学科的团队，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，快速回答以下基本问题 多药转运和外排泵的抑制。应用于Nora的所有方法都将可翻译为 其他传输系统。

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.