Chemical probes of vulnerable pathways in antibiotic-resistant pathogens

描述(申请人提供)：结核病的病原体结核分枝杆菌是一种具有全球重要性的病原体；每年报告900万新的结核病病例，其中100-200万人死亡。每年有50万例耐药结核病病例，而且抗生素耐药性问题正在上升。结核病治疗是一个漫长的过程(6-24个月)，迫切需要更新、更有效的抗生素。为了开发新药，我们需要了解哪些代谢过程对细菌的生存和致病最重要。化学实体可以成为识别支撑生存的关键细胞过程的强大探测器。然而，最近的工作主要集中在识别必需基因的遗传方法上，而在识别有效抗菌化合物靶标的途径方面进展甚微。最近，一些目标未知的新化合物被鉴定为对包括结核分枝杆菌在内的耐药病原体具有杀灭或静态活性。我们建议结合几种化合物类别应用技术组合，以识别易受攻击的途径，这些途径可以用化合物作为化学探针来彻底表征。我们将使用三种主要方法来表征抗结核药物。(1)分离复合耐药株(自发突变株或高表达重组株)，以确定耐药蛋白靶点和耐药模式。(2)我们将使用三种方法(亲和层析、光亲和标记和酵母三杂交系统)来鉴定与化合物结合的蛋白质靶标。(3)我们将观察接触化合物对细胞代谢物的影响以及对活性氧的诱导。这些方法将使我们能够确定每种化合物靶标的途径和特定蛋白质；这些靶标将构成未来药物发现和开发的基础。 公共卫生相关性：结核分枝杆菌是人类结核病的病原体，这是一种毁灭性的传染病，每年导致近200万人死亡，800多万人感染；多重耐药和极端耐药菌株的威胁越来越大。我们的目标是确定新发现的化合物如何杀死结核杆菌，并利用这些信息寻找新的药物靶点。

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. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis is the causative agent of human tuberculosis, a devastating infectious disease, which kills nearly 2 million and infects more than 8 million people each year; there is an increasing threat from multi-drug resistant and extremely drug resistant strains. We aim to determine how newly discovered compounds kill the TB bacilli and to use this information to find new drug targets.