Chemical probes of vulnerable pathways in antibiotic-resistant pathogens

描述（由申请人提供）：结核分枝杆菌是结核病（TB）的病原体，是一种具有全球重要性的病原体；每年报告的结核病新发病例为900万例，死亡人数为100万至200万。每年有50万例耐药结核病病例，抗生素耐药性问题正在加剧。结核病治疗是一个漫长的过程（6-24个月），因此迫切需要更新、更有效的抗生素。为了开发新的药物，我们需要了解哪些代谢过程对细菌的生存和发病最重要。化学实体可以是识别支撑生存的关键细胞过程的有力探针。然而，最近的工作主要集中在遗传方法来识别必需基因，在识别有效抗菌化合物靶向的途径方面进展甚微。最近，一些具有未知靶点的新化合物已被鉴定出对耐药病原体（包括结核分枝杆菌）具有杀灭或静态活性。我们建议将技术与几种化合物类别结合起来，以确定易损途径，这些易损途径可以使用化合物作为化学探针进行彻底表征。我们将使用三种主要方法来表征抗结核药物。(1)分离复合耐药菌株（自发突变株或过表达重组菌株），确定蛋白靶点和耐药模式。(2)采用亲和层析法、光亲和标记法和酵母三杂交系统三种方法鉴定与化合物结合的蛋白靶点。(3)我们将观察化合物暴露对细胞代谢物和活性氧诱导的影响。这些方法将使我们能够识别每种化合物靶向的途径和特定蛋白质；这些靶点将构成未来药物发现和开发的基础。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a pathogen of global importance; 9 million new cases of TB are reported annually, with 1-2 million deaths. There are half a million cases of drug resistant TB cases each year and the problem of antibiotic resistance is rising. TB treatment is a lengthy process (6-24 months) giving rise to an urgent need for newer, more effective antibiotics. In order to develop new drugs, we need to understand which metabolic processes are the most important for bacterial survival and pathogenesis. Chemical entities can be powerful probes for the identification of key cellular processes underpinning survival. However, most recent work has focused on genetic approaches to identifying essential genes and there has been little progress in identifying the pathways that effective anti-bacterial compounds target. Recently, a number of novel compounds with unknown targets have been identified with cidal or static activity against drug-resistant pathogens including M. tuberculosis. We propose to apply a combination of techniques in conjunction with several compound classes in order to identify vulnerable pathways, which can be thoroughly characterized using compounds as chemical probes. We will use three main approaches to characterize anti-tubercular agents. (1) We will isolate compound-resistant strains (spontaneous mutants or over-expressing recombinant strains) to identify the protein targets and mode of resistance. (2) We will use three methods (affinity chromatography, photoaffinity labeling, and yeast three hybrid system) to identify protein targets which bind to the compounds. (3) We will look at the effect of compound exposure on cell metabolites and the induction of reactive oxygen species. Together these methods will enable us to identify the pathways and specific proteins that are targeted by each compound; such targets will form the basis for future drug discovery and development.