Validating targets in p. aeruginosa type III secretion using chemical probes

验证第 14 页中的目标。

基本信息

批准号：
8452056
负责人：
Donald T Moir
金额：
$ 80.42万
依托单位：
MICROBIOTIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-03 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8452056
关键词：
Address Affect Affinity Affinity Labels Animals Anti-Bacterial Agents Antibiotic Resistance Antibiotic Therapy Antibiotics Back Bacteremia Bacteria Bacterial Toxins Binding Sites Biological Assay Biology Categories Cessation of life Chemical Structure Chemicals Clinical Complex Dependency Development Disease Drug resistance Engineering Escape Mutant Exhibits Failure Gene Targeting Genes Goals Human Immune Immune response Immune system Individual Infection Intoxication Kinetics Label Libraries Mediating Medical Methods Modeling Molecular Molecular Genetics Molecular Target Mutate Mutation Mutation Analysis Organism Outcome Pathogenicity Pathway interactions Patients Phagocytes Pharmaceutical Chemistry Pharmaceutical Preparations Pneumonia Prevalence Proteins Pseudomonas aeruginosa Publishing Recovery Regulation Research Role Site Structure Toxin Urinary tract infection Ventilator Virulence affinity labeling attributable mortality cytotoxic cytotoxicity deep sequencing drug discovery experience inhibitor/antagonist multidisciplinary mutant pathogen pathogenic bacteria protective effect resistant strain screening small molecule tool treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is the leading cause of ventilator-associated pneumonia (VAP), and current antibiotic treatment strategies 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 validating targets in the type III secretion (T3SS) pathway that are susceptible to inhibition by small molecules and determining their roles in the T3SS host-pathogen interaction. T3SS is the major P. aeruginosa virulence determinant contributing to the establishment and dissemination of infections (e.g. VAP, bacteremia, urinary tract infections). It is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes, thereby weakening the host's innate immune defenses. 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 strategy employed in this project is to use existing chemical probes to determine which components of the complex T3SS machine are susceptible to inhibition by small molecule compounds. Then, probes and strains carrying mutations in the probe targets will be used to define the roles of those vulnerable components in the host-pathogen interaction. Results will provide up to four well-validated, functionally annotated, druggable targets in the T3SS host-pathogen interaction. Four published T3SS inhibitors with unrelated chemical structures that are inhibitory to P. aeruginosa T3SS at non-cytotoxic concentrations have been selected as probes. Two of the probes are potent inhibitors of both T3SS-mediated secretion and translocation of effector toxins while the other two probes inhibit only secretion or translocation. Thus, these fou chemical probes likely inhibit at least three distinct targets or distinct regions within one or moe targets. In Aim 1, two parallel approaches will be used to identify the molecular targets of these four probes - (a) addition of photo-reactive and molecular handle moieties to permissible sites on the probes, application of photo-affinity probes to modify the target(s), recovery and identification of modified proteins; (b) application of molecular genetic tools to enrich for probe escape mutants followed by identification of the mutated gene(s) by deep sequencing. Finally, target identity will be confirmed by mutation analysis, and target gene mutant libraries will be prepared to facilitate understanding the role of each target in T3SS. In Aim 2, the probes and mutant libraries will be used to define the role of each probe target in the T3SS host-pathogen interaction. Effects of the probes and mutants on twelve distinct steps in the T3SS pathway within the broad categories of regulation, assembly, secretion, translocation, and cytotoxicity wil be used to dissect the roles of the targets in the T3SS machine and in host-pathogen interactions. Results will provide druggable, disease-relevant T3SS targets with characterized escape mutants for use in drug-discovery screening. Ideal targets will be prioritized as highly sensitive to rapid inhibition by probes and involved in critical roles in host-pathogen interaction.

描述（由申请人提供）：铜绿假单胞菌是呼吸机相关性肺炎 (VAP) 的主要原因，当前的抗生素治疗策略的失败率高达 18%，即使该生物体对所施用的抗生素敏感。该项目的目标是通过验证 III 型分泌 (T3SS) 途径中易受小分子抑制的靶标并确定它们在 T3SS 宿主-病原体相互作用中的作用来满足这一关键的医疗需求。 T3SS 是导致感染（例如 VAP、菌血症、尿路感染）建立和传播的主要铜绿假单胞菌毒力决定因素。细菌利用它分泌毒素效应物并将其转移到宿主吞噬细胞中，从而削弱宿主的先天免疫防御。功能性 T3SS 的存在与患者不良临床结果和死亡显着相关，并显着降低动物感染模型的存活率。该项目采用的策略是使用现有的化学探针来确定复杂的 T3SS 机器的哪些组件容易受到小分子化合物的抑制。然后，在探针目标中携带突变的探针和菌株将用于定义这些易受攻击的成分在宿主-病原体相互作用中的作用。结果将在 T3SS 宿主-病原体相互作用中提供多达四个经过充分验证、功能注释的可药物靶标。四种已发表的具有不相关化学结构的 T3SS 抑制剂在非细胞毒性浓度下对铜绿假单胞菌 T3SS 具有抑制作用，已被选为探针。其中两种探针是 T3SS 介导的效应毒素分泌和易位的有效抑制剂，而另两种探针仅抑制分泌或易位。因此，这些化学探针可能抑制至少三个不同的靶标或一个或多个靶标内的不同区域。在目标 1 中，将使用两种并行方法来识别这四种探针的分子靶标 - (a) 在探针上允许的位点添加光反应性和分子手柄部分，应用光亲和探针来修饰靶标（ s)、修饰蛋白的回收和鉴定； (b) 应用分子遗传学工具富集探针逃避突变体，然后通过深度测序鉴定突变基因。最后，通过突变分析确认靶点身份，并准备靶点基因突变库，以方便了解每个靶点在T3SS中的作用。在目标 2 中，探针和突变体库将用于定义每个探针靶标在 T3SS 宿主-病原体相互作用中的作用。探针和突变体对 T3SS 途径中十二个不同步骤（调节、组装、分泌、易位和细胞毒性等大类）的影响将用于剖析靶标在 T3SS 机器和宿主-病原体相互作用中的作用。结果将提供可药物化的、与疾病相关的 T3SS 靶点，以及特征性的逃逸突变体，用于药物发现筛选。理想的靶标将被优先考虑为对探针的快速抑制高度敏感，并在宿主-病原体相互作用中发挥关键作用。