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; 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.

抗抗生素的革兰氏阴性感染对人类健康构成了重大威胁。革兰氏阴性生物的一个定义特征是存在第二膜外膜（OM）的存在，该膜调节分子进入周期。 OM是针对革兰氏阳性生物有效的抗生素，例如万古霉素，即使革兰氏阴性菌包含相同的靶标，也对革兰氏阴性菌无效。 OM由内部小叶中含有磷脂的不对称双层和外部小叶中的脂多糖（LPS）组成。细胞表面上的LP会产生一个聚电解质网状网状，该网格充当了通过亲水和疏水分子传递的强大障碍。防止适当的LPS生物合成和组装通常是致命的，因为LPS在大多数革兰氏阴性生物中都是必不可少的。在有LPS组装抑制剂存在下可行的那些生物具有OM缺陷，使它们对通常无法穿透OM屏障的抗生素敏感。在这笔赠款中，我们建议开发一种涉及基于目标和细胞的筛查的综合方法，以鉴定铜绿假单胞菌和鲍曼尼杆菌杆菌中OM生物发生的小分子抑制剂，这是两种机会性病原体，多种耐药性猖rug。 AIM 1将使用基于目标的屏幕来识别LPTB的抑制剂，LPTB的抑制剂是将LPS从内膜转移到将其转移到OM的蛋白质的基本ATPase。 AIM 2将使用基于细胞的记者测定法来鉴定铜绿假单胞菌中OM生物发生的抑制剂。 AIM 3将利用与A. baumannii中OM生物发生有关的后期酶的条件必要性，以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。一种基于荧光的新型测定法，该测定法在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导致LPS组装中的缺陷。我们将验证所有目标中发现的热门化合物使用我们实验室中开发的新生物化学和微生物学方法。最有希望的命中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。使用靶基于靶标和细胞筛网的这种组合，我们希望鉴定出新的抗生素来治疗革兰氏阴性感染以及通过使OM泄漏通过临床使用抗生素增强临床使用的化合物。