Discovery of Molecules to disrupt the outer membrane of Gram-negative pathogens

发现破坏革兰氏阴性病原体外膜的分子

基本信息

批准号：
9017928
负责人：
Daniel Kahne
金额：
$ 90.31万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-03-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9017928
关键词：
ATP phosphohydrolase Acinetobacter baumannii Affect Antibiotic Resistance Antibiotics Biochemical Biogenesis Biological Assay Carrier Proteins Cell Membrane Permeability Cell surface Cells Clinical Colistin Collaborations Complex Cytoplasm Defect Enzymes Escherichia coli Fluorescence Genes Genetic Genetic Transcription Gram-Negative Bacteria Grant Health Hospitals Human In Vitro Infection Instruction Knock-out Label Laboratories Lead Lipopolysaccharide Biosynthesis Pathway Lipopolysaccharides Luciferases Membrane Methods Multi-Drug Resistance Organism Pathway interactions Phospholipids Polymyxins Power Sources Proteins Pseudomonas aeruginosa Reporter Reporter Genes Reporting Research Personnel Services Specificity Staging Surface Testing Vancomycin antimicrobial bacterial resistance base cell envelope design genetic approach high throughput screening in vivo inhibitor/antagonist innovation killings membrane biogenesis mortality mutant novel pathogen periplasm prevent promoter proteoliposomes reconstitution research study screening small molecule small molecule inhibitor therapy development vaccine development

项目摘要

Antibiotic-resistant Gram-negative infections pose a major threat to human health. A defining feature of Gram-negative organisms is the presence of a second membrane, the outer membrane (OM), which regulates access of molecules to the periplasm. The OM is the reason that antibiotics that are effective against Gram-positive organisms, such as vancomycin, are not effective against Gram-negatives even though Gram-negatives contain the same targets. The OM is composed of an asymmetric bilayer containing phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS on the cell surface creates a polyelectrolyte mesh that acts as a formidable barrier to passage of both hydrophilic and hydrophobic molecules. Preventing proper LPS biosynthesis and assembly is often lethal since LPS is essential in most Gram-negative organisms. Those organisms that are viable in the presence of LPS assembly inhibitors have OM defects that render them sensitive to antibiotics that cannot normally penetrate the OM barrier. In this grant, we propose to develop a comprehensive approach involving both target- and cell-based screens to identify small molecule inhibitors of OM biogenesis in Pseudomonas aeruginosa and Acinetobacter baumannii, two opportunistic pathogens for which multi-drug resistance is rampant. Aim 1 will use a target-based screen to identify inhibitors of LptB, the essential ATPase that powers the transfer of LPS from the inner membrane to proteins that translocate it to the OM. Aim 2 will use cell-based reporter assays to identify inhibitors of OM biogenesis in P. aeruginosa. Aim 3 will exploit the conditional essentiality of late stage enzymes involved in OM biogenesis in A. baumannii to develop a cell-based, pathway-speciflc screen to discover small-molecule inhibitors of LPS biogenesis. A novel fluorescence-based assay that reports on properly assembled LPS on the cell surface will be used to show that inhibitors found in the pathway-specific screen lead to defects in LPS assembly. We will validate that the hit compounds found in all aims are on target using novel biochemical and microbiological approaches developed in our labs. The most promising hit compounds will be subjected to optimization and in vivo efficacy studies in collaboration with the Discovery and Translational Services (DTS) Core. Using this combination of target- and cell-based screens we hope to identify new antibiotics to treat Gram-negative infections as well as compounds that potentiate clinically used antibiotics by rendering the OM leaky.

抗生素耐药性革兰氏阴性菌感染对人类健康构成重大威胁。革兰氏阴性生物体的一个决定性特征是存在第二层膜，即外膜（OM），它调节分子进入周质。 OM 是对革兰氏阳性菌有效的抗生素（如万古霉素）对革兰氏阴性菌无效的原因，即使革兰氏阴性菌含有相同的靶标。 OM 由不对称双层组成，内层含有磷脂，外层含有脂多糖 (LPS)。细胞表面的脂多糖形成聚电解质网，作为亲水性和疏水性分子通过的强大屏障。由于 LPS 在大多数革兰氏阴性生物体中至关重要，因此阻止适当的 LPS 生物合成和组装通常是致命的。那些在 LPS 组装抑制剂存在下仍能存活的生物体具有 OM 缺陷，这使得它们对通常无法穿透 OM 屏障的抗生素敏感。在这笔资助中，我们建议开发一种综合方法，涉及基于靶标和基于细胞的筛选，以识别铜绿假单胞菌和鲍曼不动杆菌（两种多药耐药性猖獗的机会性病原体）中 OM 生物发生的小分子抑制剂。目标 1 将使用基于靶标的筛选来识别 LptB 抑制剂，LptB 是一种必需的 ATP 酶，为 LPS 从内膜转移到蛋白质（将其易位到 OM）提供动力。目标 2 将使用基于细胞的报告基因检测来鉴定铜绿假单胞菌中 OM 生物发生的抑制剂。目标 3 将利用鲍曼不动杆菌中 OM 生物发生所涉及的后期酶的条件必要性来开发基于细胞的途径特异性筛选，以发现 LPS 生物发生的小分子抑制剂。一种新的基于荧光的检测方法报告了细胞表面上正确组装的 LPS，将用于表明在途径特异性筛选中发现的抑制剂会导致 LPS 组装缺陷。我们将使用我们实验室开发的新型生化和微生物方法来验证所有目标中发现的命中化合物是否达到目标。最有前途的热门化合物将与发现和转化服务 (DTS) 核心合作进行优化和体内功效研究。通过使用基于靶标和细胞的筛选的组合，我们希望找到治疗革兰氏阴性菌感染的新抗生素，以及通过使 OM 渗漏来增强临床使用抗生素的化合物。