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机械通气患者院内获得性肺炎的主要原因。目前的抗生素治疗显示出高达18%的失败率，即使当生物体对所施用的抗生素敏感时也是如此。该项目的目标是通过鉴定3型分泌系统（T3SS）的特定抑制剂并将其开发成针对铜绿假单胞菌的新型治疗剂来解决这一关键的医疗需求。T3SS是铜绿假单胞菌感染建立和传播的主要毒力因子，细菌利用T3SS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反应。在动物感染模型中，功能性T3SS的存在与不良临床结果和患者死亡显著相关，并显著降低生存率。在本项目中开发的T3SS抑制剂将与抗假单胞菌药物联合治疗和预防，以抑制T3SS介导的吞噬细胞中毒，从而增强强大的宿主先天免疫反应，并增强共给药抗生素的活性。在I期研究中，我们发现了3种不同化学型的15种新型P. aeruginosa T3SS抑制剂，具有以下特性，有利于进一步开发：(a)新颖的，化学上可处理的结构，(b)任何报道的P. aeruginosa T3SS抑制剂的最高效价（IC50=1-2 5g/ml）， (c)选择性指数（CC50/IC50） bb0 50, (d)有利的初步构效关系（SAR），包括严格的立体特异性活性，明确定义立体中心取代基大小，以及在支架的三个区域鉴定出五种修饰，以增加效价；及(e)不受铜绿假单胞菌外排的影响。我们在II期的策略是优化这些最有前途的结构作为临床前候选药物。将应用生物化学和分子遗传学方法来确定这些抑制剂的分子靶点和抗性突变的频率。在毒性和药代动力学评估之后，抑制剂将在铜绿假单胞菌感染、急性肺炎和菌血症的两种小鼠模型中作为单药和与抗假单胞菌药头孢他啶联合进行疗效测试。该提案的主要里程碑是选择抗t3ss临床前候选药物，这些候选药物将进入研究新药（IND），从而在该项目的第三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)基于构效关系合成结构多样的苯氧乙酰胺系列类似物；(2)根据T3SS抑制活性的效力和选择性以及有利的ADME特性对类似物进行排序；(3)确定苯氧乙酰胺T3SS抑制剂系列的分子靶点及耐药频率；(4)在动物模型中测定铅化合物的急性毒性、药代动力学参数和功效。

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. 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.