Type III Secretion Inhibitors for Anti-Infective Therapy

描述(申请人提供)：铜绿假单胞菌感染是机械通气患者医院获得性肺炎的主要原因。目前的抗生素治疗失败率高达18%，即使当机体对正在使用的抗生素敏感时也是如此。该项目的目标是通过确定三型分泌系统(T3SS)的特定抑制剂并将它们开发成对抗铜绿假单胞菌的新型治疗剂来满足这一关键的医疗需求。T3SS是导致铜绿假单胞菌感染的主要毒力因子，被细菌利用来分泌毒素效应物并将其转运到宿主吞噬细胞中，从而削弱宿主的天然免疫反应。在动物感染模型中，功能性T3SS的存在与患者不良的临床结局和死亡显著相关，并显著降低存活率。本项目中开发的T3SS抑制剂将与抗假单胞菌药物联合用于治疗和预防，以抑制T3SS介导的吞噬细胞中毒，从而增强强大的宿主先天性免疫反应，并增强联合使用抗生素的活性。在第一阶段，我们在3种不同的化学类型中发现了15种新的铜绿假单胞菌T3SS抑制剂，以下特性有利于进一步的开发：(A)新的、化学上易于处理的结构，(B)任何已报道的铜绿假单胞菌T3SS抑制剂的最高效力(IC50=1-2.5g/ml)，(C)选择性指数(CC50/IC50)和GT；50，(D)良好的构效关系(SAR)，包括严格的立体专一性，立体中心取代基大小的明确定义，以及在支架的三个区域识别五种提高效力的修饰；和(E)不受铜绿假单胞菌外排的影响。我们在第二阶段的战略是优化这些结构中最有希望的临床前候选结构。生化和分子遗传学方法将被用来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之后，将测试抑制剂作为单一药物以及与抗假单胞菌药物头孢他啶联合使用在两种铜绿假单胞菌感染、急性肺炎和菌血症小鼠模型中的疗效。这项建议的主要里程碑是选择抗T3SS临床前候选药物，这将推进到研究新药(IND)，使毒理学和安全药理学研究在该项目的第三阶段。我们将完成以下具体目标：(1)根据构效关系合成结构多样化的苯氧乙酰胺Hit系列类似物；(2)根据T3SS抑制活性的效力和选择性以及良好的ADME特性对类似物进行优先排序；(3)确定苯氧乙酰胺T3SS抑制剂系列的分子靶点和耐药频率；(4)在动物模型中确定先导化合物的急性毒性、药代动力学参数和疗效。

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.