Type III Secretion Inhibitors for Anti-Infective Therapy

描述(申请人提供)：铜绿假单胞菌是一种常见且极其致命的原因，可导致免疫功能低下/抑制的患者(如艾滋病毒和癌症)、囊性纤维化患者、机械通气者或有烧伤创面的患者发生严重感染。频繁的抗生素耐药性和铜绿假单胞菌的高度毒力使其比大多数其他细菌更致命。作用于新的可接触靶点的新的抗生素化学类别对于持续有效地治疗铜绿假单胞菌至关重要，因为此类药物将不受现有耐药机制的影响。该项目的战略是通过筛选一系列不同的合成和天然产物化合物来对抗对毒力至关重要的细胞外靶点，从而开发新药。III型分泌系统(TTSS)是铜绿假单胞菌重要的临床靶点，主要负责蛋白毒素(效应物)的分泌和转位。针对TTSS的药物可以与杀菌抗生素联合用于免疫功能低下的患者，它们本身可能对免疫系统正常的患者有用或用于预防性应用。该项目的目标是确定TTSS的特定抑制剂，并将其开发为治疗铜绿假单胞菌的新型抗生素。在第一阶段，将开发两种新的铜绿假单胞菌TTSS全细胞报告分析方法。高通量筛选的初步试验将通过TTSS检测效应器-B-内酰胺酶嵌合报告的分泌和移位抑制物进入CHO细胞。第二个实验将基于TTSS基因表达和分泌通道活性之间的紧密联系，通过构建转录荧光素酶报告，当TTSS功能丧失时，该报告下调100倍。将用初级试验筛选不同的化学库，通过转录报告试验和TTSS依赖的细胞毒性试验验证HITS。验证的命中将通过展示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抑制的光谱来优先考虑。我们将完成以下特定目标：(1)建立高通量分泌型报告筛选铜绿假单胞菌TTSS抑制剂；(2)建立转录报告二级检测铜绿假单胞菌TTSS抑制剂；(3)筛选多样化的化合物文库以识别和验证TTSS抑制剂；以及(4)优先考虑体外有效性、机制、光谱和选择性的筛选。

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, 2 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-B-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.