Type III Secretion Inhibitors for Anti-Infective Therapy

描述（由申请人提供）：铜绿假单胞菌是免疫功能低下/抑制患者（例如艾滋病毒和癌症）、囊性纤维化患者、机械通气或烧伤创面患者严重感染的常见且极其致命的原因。铜绿假单胞菌频繁的抗生素耐药性和高毒性使其比大多数其他细菌更致命。新的化学类抗生素作用于新的可获得的靶点，对于持续有效治疗铜绿假单胞菌至关重要，因为这些药物将不受现有耐药机制的影响。该项目的策略是通过筛选多种合成和天然产物化合物来开发新药，以对抗对毒性至关重要的细胞外靶点。III型分泌系统（TTSS）专门分泌蛋白毒素（“效应器”）并将其转运到人类细胞的细胞质中，已被证实是铜绿假单胞菌的临床重要靶点。针对TTSS的药物可与抗菌抗生素联合用于免疫功能低下的患者，它们可能对免疫系统正常的患者单独有用或用于预防应用。本项目的目标是鉴定特异性的TTSS抑制剂，并将其开发成治疗铜绿假单胞菌的新型抗生素。在第一阶段，两种新的全细胞报告检测方法将用于铜绿假单胞菌的TTSS。高通量筛选的主要试验将检测通过TTSS进入CHO细胞的效应内酰胺酶嵌合报告基因的分泌抑制剂和易位。第二项试验将基于TTSS基因表达与分泌通道活性之间的紧密联系，通过构建一个转录荧光素酶报告基因，该报告基因在TTSS功能丧失时下调100倍。不同的化学文库将通过初级试验筛选，通过转录报告试验和ttss依赖性细胞毒性试验验证。通过证明对细菌细胞生长和哺乳动物细胞活力的最小抑制，对细胞膜的最小破坏，以及通过确定TTSS抑制的光谱，验证命中将被优先考虑。我们将实现以下具体目标：(1)开发铜绿假单胞菌TTSS抑制剂的高通量分泌报告基因筛选；(2)建立铜绿假单胞菌TTSS抑制剂的转录报告二级检测方法；(3)筛选多种化合物文库，鉴定和验证TTSS抑制剂；(4)对体外效价、机制、谱和选择性进行优先筛选。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is a common and extremely virulent cause of serious infections in immune- compromised/suppressed patients (e.g., HIV and cancer), cystic fibrosis patients, and those on mechanical ventilation or with burn wounds. Frequent antibiotic resistance and the highly virulent nature of P. aeruginosa make it deadlier than most other bacteria. New chemical classes of antibiotics acting on novel accessible targets are crucial for continued effective therapy against P. aeruginosa because such drugs will not be subject to existing resistance mechanisms. The strategy of this project is to develop new drugs by screening a diverse collection of synthetic and natural product compounds against extra-cellular targets that are critical for virulence. The type III secretion system (TTSS), dedicated to the secretion of protein toxins ("effectors") and their translocation into the cytoplasm of human cells has been validated as a clinically important target in P. aeruginosa. Drugs targeting TTSS may be used in combination with bactericidal antibiotics in immune-compromised patients, and they may be useful on their own in patients with competent immune systems or for prophylactic application. The goal of this project is to identify specific inhibitors of TTSS and to develop them into novel antibiotics for therapy against P. aeruginosa. In Phase I, two new whole-cell reporter assays for the TTSS of P. aeruginosa will be developed. The primary assay for high throughput screening will detect inhibitors of secretion and translocation of an effector-¿-lactamase chimeric reporter via TTSS into CHO cells. The second assay will be based on the tight linkage between TTSS gene expression and the activity of the secretion channel by building a transcriptional luciferase reporter which is down-regulated by 100-fold upon loss of TTSS function. A diverse chemical library will be screened with the primary assay, hits validated by the transcriptional reporter assay as well as by a TTSS-dependent cytotoxicity assay. Validated hits will be prioritized by demonstrating minimal inhibition of both bacterial cell growth and mammalian cell viability, minimal disruption of cell membranes, and by determining the spectrum of TTSS inhibition. We will accomplish the following specific aims: (1) Develop a high-throughput secreted reporter screen for inhibitors of P. aeruginosa TTSS; (2) Develop a transcriptional reporter secondary assay for inhibitors of P. aeruginosa TTSS; (3) Screen a diverse compound library to identify and validate TTSS inhibitors; and (4) Prioritize validated screening hits for in vitro potency, mechanism, spectrum, and selectivity.