Development of validated probes for the bacterial type III secretion system

随着抗生素耐药性的增加，针对细菌病原体的新疗法的开发 对全球公共卫生至关重要。细菌III型分泌系统(T3SS)代表了一种极好的药物 靶标是因为它可以从外部被小分子接触，并使假单胞菌具有毒力， 沙门氏菌、衣原体和许多其他重要病原体。我们已经开发出一种高产量的 筛选发现T3SS抑制剂的渠道，并通过试点展示了我们方法的健壮性 筛选鉴定对T3SS有活性的三类化合物。我们现在建议扩大我们的 范围和筛选三个独特的文库，包括成员开发的约58,000个天然产品馏分 以及两个商业合成化学库。根据疾控中心的数据，每年 美国发生了50,000多起与医疗保健相关的铜绿假单胞菌感染，其中6,000起 由多重耐药菌株引起。假单胞菌T3SS抑制剂的鉴定及应用前景 治疗方面，我们将实现以下目标。在目标1中，我们将实现我们的主屏幕和 用于识别具有特定T3SS抑制作用的天然产物组分和合成化合物的反筛选 活动。在目标2中，我们将使用三种正交的不同方法来验证目标1中识别的命中。这个 将确定生物活性天然产物的特性和结构，并进行初步结构活性分析 在确定的合成脚手架上进行。将对优先化合物进行提纯或合成并进行评估 对于非目标活性，如果有的话，以及在多个相关病原体中对T3SS的活性广度。在AIM 3.将使用并行的遗传和生物化学方法确定行动模式。这份严谨 战略将提供约10个T3SS抑制剂化学探针，以确定对P. 铜绿假单胞菌和潜在的其他病原体。

With increasing incidence of antibiotic resistance, development of new therapies against bacterial pathogens is essential for global public health. The bacterial type III secretion system (T3SS) represents an excellent drug target because it is externally accessible to small molecules and enables virulence of Pseudomonas, Salmonella, Chlamydia, and numerous other important pathogens. We have developed a high throughput screening pipeline to discover T3SS inhibitors and have shown the robustness of our approach through pilot screens identifying three classes of compounds active against the T3SS. We now propose to broaden our scope and screen three unique libraries comprising ~58,000 natural product fractions developed by members of our consortium as well as two commercial synthetic chemical libraries. According to the CDC, every year over 50,000 healthcare-associated Pseudomonas aeruginosa infections occur in the U.S., >6,000 of which are caused by multidrug resistant strains. To identify Pseudomonas T3SS inhibitors and potential future therapeutics, we will carry out the following aims. In Aim 1, we will implement our primary screen and counterscreens to identify natural product fractions and synthetic compounds with specific T3SS inhibitory activity. In Aim 2, we will validate hits identified in Aim 1, using three orthogonal distinct approaches. The identity and structure of bioactive natural products will be determined and initial structure activity analysis performed on identified synthetic scaffolds. Prioritized compounds will be purified or synthesized and evaluated for off target activity, if any, as well as breadth of activity against T3SSs in multiple relevant pathogens. In Aim 3, mode of action will be determined, using parallel genetic and biochemical approaches. This rigorous strategy will provide ~10 T3SS inhibitor chemical probes with identified molecular targets active against P. aeruginosa and potentially other pathogens.