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High-throughput fluorescence screening for inhibitors of TonB-dependent iron transport

高通量荧光筛选 TonB 依赖性铁转运抑制剂

基本信息

批准号：
8969952
负责人：
PHILLIP E KLEBBA
金额：
$ 23.75万
依托单位：
KANSAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8969952
关键词：
2,4-Dinitrophenol Animals Antibiotics Assimilations Bacteria Bacterial Infections Bacteriophages Binding Binding Sites Biochemical Biological Assay Biological Factors Cell surface Cells Chemicals Collection Complex Development Dinitrophenols Enterobacter aerogenes bacterium Enterobactin Escherichia coli FDA approved Ferrichrome Fluorescein Fluorescence Fluorescence Spectroscopy Fluorescent Probes Genetic Engineering Gram-Negative Bacteria Growth Haemophilus influenzae Human Hydrazones Individual Iron Kansas Klebsiella pneumonia bacterium Knowledge Label Laboratories Lead Libraries Life Location Maleimides Measurement Membrane Membrane Proteins Membrane Transport Proteins Metabolism Metals Methods Microbe Molecular Monitor Motion Nature Neisseria meningitidis Pathogenesis Pathogenicity Pathway interactions Peptidoglycan Performance Physiology Preparation Procedures Process Proton-Motive Force Pseudomonas aeruginosa Reaction Research Salmonella typhi Salmonella typhimurium Siderophores Site Staging Structure Surface Temperature Testing Text Time Transport Reaction Universities Validation Vibrio cholerae Vitamin B 12 aerobactin bacteriocin base biophysical analysis cell envelope cell preparation fluorophore high throughput screening in vivo inhibitor/antagonist insight killings mesoxalonitrile microbial novel pathogen permease public health relevance receptor research study scaffold screening uptake vibriobactin

项目摘要

DESCRIPTION (provided by applicant): The ability of microbes to acquire iron is a key aspect of microbial invasiveness and pathogenicity. TonB-dependent transporters in the Gram-negative bacterial outer membrane catalyze the uptake of iron complexes called ferric siderophores, and the assimilation of these metal chelates is a determinant of the pathogenesis of many species, including Escherichia coli, Salmonella typhi, Neisseria meningitidis, Vibrio cholerae and others. Unfortunately, the full molecular understanding of these transport reactions is lacking, which impairs our ability to conceive of potential inhibitors. Nevertheless, it's known that the E. coli outer membrane protein FepA accomplishes active iron transport by a process that involves extensive conformational motion. The proposed research capitalizes on this knowledge with a fluorescence spectroscopic assay that monitors uptake of the siderophore ferric enterobactin. The experiments will encompass high-throughput screening of validation and primary libraries of compounds at the University of Kansas High-Throughput Screening facility. The three libraries include standards and test chemicals that total ~17,500 unique molecules. These tests, performed in 384-well microtiter plates, will identify compounds that inhibit TonB-dependent iron transport by Gram- negative bacteria, creating a panel of candidates for further testing as antibiotics against bacterial pathogenesis.

描述（由申请人提供）：微生物获取铁的能力是微生物侵袭性和致病性的一个关键方面。革兰氏阴性细菌外膜中的 TonB 依赖性转运蛋白催化被称为三价铁铁载体的铁复合物的摄取，这些金属螯合物的同化是许多物种发病机制的决定因素，包括大肠杆菌、伤寒沙门氏菌、脑膜炎奈瑟氏菌、霍乱弧菌等。不幸的是，缺乏对这些转运反应的完整分子理解，这削弱了我们构思潜在抑制剂的能力。然而，众所周知，大肠杆菌外膜蛋白 FepA 通过涉及广泛构象运动的过程来实现主动铁转运。拟议的研究利用了这一知识，通过荧光光谱测定来监测铁载体铁肠杆菌素的摄取。这些实验将包括在堪萨斯大学高通量筛选设施中对化合物的验证和初级文库进行高通量筛选。这三个库包含总共约 17,500 个独特分子的标准品和测试化学品。这些测试在 384 孔微量滴定板上进行，将鉴定出抑制革兰氏阴性菌依赖 TonB 的铁转运的化合物，从而创建一组候选药物，用于进一步测试作为针对细菌发病机制的抗生素。