Development of validated probes for the bacterial type III secretion system

随着抗生素耐药性发生率的增加，开发针对细菌病原体的新疗法是必要的。 对全球公共卫生至关重要。细菌III型分泌系统（T3 SS）是一种优秀的药物 靶向，因为它是外部可接近的小分子，并使假单胞菌的毒力， 沙门氏菌，衣原体，和许多其他重要的病原体。我们开发了一种高通量 筛选管道，以发现T3 SS抑制剂，并通过试点显示了我们方法的稳健性 筛选鉴定了三类对T3 SS有活性的化合物。我们现在建议扩大我们的 范围和筛选三个独特的图书馆，其中包括成员开发的约58，000种天然产物馏分 以及两个商业合成化学库。根据疾病预防控制中心的数据， 在美国发生了超过50，000例与卫生保健相关的铜绿假单胞菌感染，>6,000人 由多重耐药菌株引起。鉴定假单胞菌T3 SS抑制剂和潜在的未来 治疗，我们将实现以下目标。在目标1中，我们将实现主屏幕， 用于鉴定具有特异性T3 SS抑制活性的天然产物级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验证目标1中确定的命中。的 确定生物活性天然产物的身份和结构，并进行初步结构活性分析 在已鉴定的合成支架上进行。优先化合物将被纯化或合成并评估 对于脱靶活性（如果有的话）以及针对多种相关病原体中的T3 SS的活性广度。在Aim中 3、将使用平行的遗传学和生物化学方法确定作用模式。这种严格 该策略将提供约10种T3 SS抑制剂化学探针，其具有抗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.