Virulence inhibitors as candidate therapeutics in CF

描述（由申请人提供）：囊性纤维化患者发病和死亡的主要原因是慢性呼吸道感染，主要由机会致病菌铜绿假单胞菌引起。在CF患者呼吸道定植的细菌利用毒力因子的武器来克服主要针对多形核白细胞的宿主防御机制。其中一种机制是产生细胞毒性和抗吞噬因子（“效应因子”），通过III型分泌系统（TTSS）靶向进入宿主细胞。本应用的目的是利用化学遗传学的新领域来开发小分子作为TTSS功能的探针。本项目的目的是利用灵敏的高通量生物测定（TTSS依赖性杀死培养的CHO细胞）来鉴定细胞毒性的小分子抑制剂，并进一步确定抑制TTSS特定阶段表达的机制，P. aeruginosa的效应物分泌及其在哺乳动物细胞中的转运。将从各种临床来源中筛选干扰TTTS作用的活性化合物，重点是粘液样和非粘液样CF分离株。此外，对于每个化合物或化合物家族，我们将确定TTSS的特定成分，这是抑制的目标。毫无疑问，迫切需要开发在CF患者呼吸道定植的所有阶段对铜绿假单胞菌有活性的新型抗菌药物。该项目的另一个成果将是鉴定小分子群并建立它们之间的结构活性关系，以便它们可以通过抑制关键的毒力机制作为先导化合物开发针对铜绿假单胞菌的治疗剂。因此，该项目可以为TTSS领域的一个新的研究方向奠定基础，从而更好地理解这一重要的效应物靶向机制，这是目前通过更传统的TTSS成分突变体的使用无法实现的。

DESCRIPTION (provided by applicant): The major cause of morbidity and mortality of patients with cystic fibrosis is the chronic respiratory infection caused primarily by an opportunistic pathogen Pseudomonas aeruginosa. The bacteria that colonize the respiratory tract of CF patients utilize an armament of virulence factors to overcome the host defense mechanisms directed primarily at polymorphonucelar leukocytes. One such mechanism is production of cytotoxic and anti-phagocytic factors ("effectors"), targeted into the host cells by the type III secretion system (TTSS). The objective of this application is to utilize the new field of chemical genetics to develop small molecules as probes of function of the TTSS. The aims of this project are to utilize a sensitive high throughput bioassay (TTSS dependent killing of cultured CHO cells) to identify small molecule inhibitors of cytotoxicity, and further define the mechanism of inhibition of the specific stages of TTSS expression, effector secretion from P. aeruginosa and their translocation into the mammalian cell. The active compounds that interfere with the action of TTTS will be screened for activity in P. aeruginosa from a variety of clinical sources, with emphasis on mucoid and non-mucoid CF isolates. Furthermore, for each compound or family of compounds, we will identify the specific component of the TTSS, which is the target of inhibition. There is little doubt that there is an urgent need to develop new classes of antimicrobials active against P. aeruginosa during all stages of respiratory track colonization of CF patients. An additional outcome of this project will be the identification of groups of small molecules and the establishment of structure activity relationships among them such that they may serve as lead compounds for the development of therapeutic agents against P. aeruginosa by inhibiting a key virulence mechanism. This project therefore can lay the foundation for a new research direction in the field of TTSS, leading to a better understanding of this important mechanism of effector targeting, which is currently not possible through the more traditional use of mutants in the TTSS components.