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

耐药的革兰氏阴性菌感染对人类健康构成了重大威胁。革兰氏阴性菌的一个明显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，即使革兰氏阴性菌含有相同的靶点，但对革兰氏阴性菌无效的原因。OM由一个不对称的双层组成，内叶含有磷脂，外叶含有脂多糖。细胞表面的脂多糖形成了聚电解质网状物，对亲水和疏水分子的通过起到了强大的屏障作用。阻止内毒素的生物合成和组装通常是致命的，因为内毒素在大多数革兰氏阴性生物中是必不可少的。那些在脂多糖组装抑制剂存在下仍能存活的微生物存在OM缺陷，这使得它们对抗生素敏感，而抗生素通常无法穿透OM屏障。在这项拨款中，我们建议开发一种综合的方法，包括基于靶点和基于细胞的筛选，以确定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，这两种条件致病菌对多药耐药非常猖獗。AIM 1将使用基于靶点的筛选来识别LptB的抑制剂，LptB是一种基本的ATPase，它推动内膜向蛋白质的转移，从而将其转移到OM。AIM 2将使用基于细胞的报告分析来确定铜绿假单胞菌OM生物发生的抑制剂。目的3将利用参与鲍曼不动杆菌OM生物发生的晚期酶的条件性重要性，开发一种基于细胞的、途径特异性的筛选，以发现内毒素生物发生的小分子抑制物。一种新的基于荧光的分析报告了细胞表面正确组装的内毒素，将用于显示在途径特异性筛选中发现的抑制物导致内毒素组装的缺陷。我们将使用我们实验室开发的新的生化和微生物方法来验证在所有AIMS中发现的命中化合物是否达到了目标。最有希望的热门化合物将与发现和翻译服务(DTS)核心合作进行优化和体内疗效研究。利用这种靶向和基于细胞的筛选的组合，我们希望识别出治疗革兰氏阴性感染的新抗生素，以及通过使OM渗漏来加强临床使用的抗生素的化合物。

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.