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

• 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
• 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

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.

耐药革兰氏阴性菌感染对人类健康构成重大威胁。革兰氏阴性菌的一个决定性特征是存在第二层膜，即外膜（OM），它调节分子进入外周质。这就是为什么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，尽管革兰氏阴性菌含有相同的目标。OM是由一个不对称的双分子层组成的，其内小叶含有磷脂，外小叶含有脂多糖（LPS）。细胞表面的LPS形成了一个聚电解质网，作为一个强大的屏障，阻止亲水性和疏水性分子通过。阻止适当的脂多糖生物合成和组装通常是致命的，因为脂多糖在大多数革兰氏阴性菌中是必需的。那些在LPS组装抑制剂存在下存活的生物体具有OM缺陷，使它们对通常不能穿透OM屏障的抗生素敏感。在这项资助中，我们建议开发一种综合的方法，包括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中OM生物发生的小分子抑制剂，这两种机会性病原体的多药耐药性非常猖獗。Aim 1将使用基于靶标的筛选来识别LptB的抑制剂，LptB是一种必需的atp酶，它驱动LPS从内膜转移到蛋白质，并将其转运到OM。目的2将使用基于细胞的报告者试验来鉴定铜绿假单胞菌中OM生物发生的抑制剂。目的3将利用鲍曼不动杆菌中参与脂多糖生物发生的后期酶的条件必要性，开发一种基于细胞的、途径特异性筛选，以发现脂多糖生物发生的小分子抑制剂。一种新的基于荧光的分析报告了细胞表面上正确组装的LPS，将用于显示在途径特异性筛选中发现的抑制剂导致LPS组装缺陷。我们将使用实验室开发的新型生化和微生物学方法验证在所有目标中发现的命中化合物。最有希望的hit化合物将与发现和转化服务（DTS） Core合作进行优化和体内疗效研究。利用这种基于靶标和基于细胞的筛选相结合，我们希望找到治疗革兰氏阴性感染的新抗生素，以及通过使OM泄漏而增强临床使用抗生素的化合物。