Type III Secretion Inhibitors for Anti-Infective Therapy

描述（由申请人提供）：铜绿假单胞菌感染是接受机械通气患者医院获得性肺炎的主要原因。目前的抗生素治疗表现出高达18%的失败率，即使当生物体对所施用的抗生素敏感时。该项目的目标是通过鉴定第三型分泌系统（T3 SS）的特异性抑制剂并将其开发为针对铜绿假单胞菌的新型治疗药物来解决这一关键的医疗需求。T3 SS是促成铜绿假单胞菌感染的建立和传播的主要毒力因子，并且被细菌利用以分泌毒素效应物并将其转运到宿主吞噬细胞中，从而削弱宿主的先天免疫应答。功能性T3 SS的存在与患者的不良临床结局和死亡显著相关，并显著降低动物感染模型的存活率。本项目中开发的T3 SS抑制剂将与抗假单胞菌药物联合治疗和预防性给药，以抑制T3 SS介导的吞噬细胞中毒，从而增强强大的宿主先天免疫应答并增强联合给药抗生素的活性。在第一阶段，我们发现了15种新的铜绿假单胞菌T3 SS抑制剂，具有3种不同的化学型，具有以下特性，有利于进一步开发：（a）新颖的、化学上易处理的结构，（B）任何报道的铜绿假单胞菌T3 SS抑制剂的最高效力（IC 50 =1-2 5g/ml），（c）选择性指数（CC 50/IC 50）>50，（d）有利的初步结构-活性关系（SAR），包括活性的严格立体特异性，立体中心处取代基大小的明确定义，和鉴定支架的三个区域中增加效力的五种修饰;和（e）在铜绿假单胞菌中不发生外排。我们在第二阶段的策略是优化这些结构中最有前途的作为临床前候选物。生物化学和分子遗传学方法将被应用于确定这些抑制剂的分子靶点和耐药突变的频率。在毒性和药代动力学评估后，将在铜绿假单胞菌感染、急性肺炎和菌血症的两种鼠模型中测试抑制剂作为单药和与抗假单胞菌剂头孢他啶组合的功效。该提案的主要里程碑是选择抗T3 SS临床前候选药物，这些候选药物将进入试验性新药（IND），从而能够在本项目的III期进行毒理学和安全药理学研究。我们将完成以下具体目标：（1）基于结构-活性关系合成苯氧基乙酰胺命中系列的结构多样的类似物;（2）根据T3 SS抑制活性的效力和选择性以及有利的ADME性质来优先考虑类似物;（3）鉴定苯氧基乙酰胺T3 SS抑制剂系列的分子靶标和抗性频率;（4）确定苯氧基乙酰胺T3 SS抑制剂系列的分子靶标。（4）在动物模型中测定先导化合物的急性毒性、药代动力学参数和功效。 公共卫生相关性：细菌病原体耐药菌株的流行率不断增加，这是一种未满足的医疗需求。第三型分泌是许多病原菌用于增加其在人类感染中的毒力的机制。通过这种方法分泌的细菌毒素降低了受感染个体自身先天免疫系统的保护作用。成功开发针对这种毒力机制的抑制剂将为对抗肺炎和菌血症等急性感染提供新武器。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa infection is the leading cause of hospital-acquired pneumonia in patients undergoing mechanical ventilation. Current antibiotic treatments exhibit failure rates as high as 18%, even when the organism is susceptible to the antibiotic being administered. The goal of this project is to address this critical medical need by identifying specific inhibitors of the type-three secretion system (T3SS) and developing them into novel therapeutic agents against P. aeruginosa. T3SS is the major virulence factor contributing to the establishment and dissemination of P. aeruginosa infections and is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes, thereby weakening the host's innate immune response. The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models. The T3SS inhibitors developed in this project will be administered therapeutically and prophylactically in combination with anti-pseudomonal agents to inhibit the T3SS-mediated intoxication of phagocytes and thereby potentiate a robust host innate immune response and enhance the activity of co-administered antibiotics. In Phase I, we discovered 15 novel inhibitors of P. aeruginosa T3SS in 3 different chemotypes, with the following properties propitious for further development: (a) novel, chemically tractable structures, (b) highest potency of any reported P. aeruginosa T3SS inhibitors (IC50=1-2 5g/ml), (c) selectivity indices (CC50/IC50) >50, (d) favorable preliminary structure-activity relationships (SAR), including strict stereo-specificity of activity, clear definition of substituent size at the stereocenter, and identification of five modifications in three regions of the scaffold that increase potency; and (e) not subject to efflux in P. aeruginosa. Our strategy in Phase II is to optimize the most promising of these structures as preclinical candidates. Biochemical and molecular genetic approaches will be applied to identify the molecular target of these inhibitors and the frequency of mutation to resistance. Following toxicity and pharmacokinetic assessment, inhibitors will be tested for efficacy as single agents and in combination with anti-pseudomonal agent ceftazidime in two murine models of P. aeruginosa infection, acute pneumonia and bacteremia. The major milestone of this proposal is to select anti-T3SS pre-clinical candidates, which will be advanced to Investigational New Drug (IND) enabling toxicology and safety pharmacology studies in Phase III of this project. We will accomplish the following specific aims: (1) synthesize structurally diverse analogs of the phenoxyacetamide hit series based on structure-activity relationships; (2) prioritize analogs by potency and selectivity of T3SS inhibitory activity, as well as favorable ADME properties; (3) identify the molecular target of the phenoxyacetamide T3SS inhibitor series and the frequency of resistance; (4) determine acute toxicity, pharmacokinetic parameters, and efficacy of lead compounds in animal models. PUBLIC HEALTH RELEVANCE: The increasing prevalence of antibiotic-resistant strains of bacterial pathogens represents an unmet medical need. Type-three secretion is a mechanism used by many pathogenic bacteria to increase their virulence in human infections. Bacterial toxins secreted by this method reduce the protective effect of the infected individual's own innate immune system. Successful development of an inhibitor targeting this virulence mechanism will provide a new weapon to combat acute infections such as pneumonia and bacteremia.