基于结核分枝菌P450酶系的新药筛选策略

结核病的控制是一项世界性难题，寻找新型抗结核药物靶标是发现抗结核病新药的关键策略之一。近年的研究发现，结核分枝杆菌（Mtb）P450酶系在脂质代谢与合成、胆固醇利用、呼吸链的电子传递等方面均扮演十分重要角色，部分亚型可作为有效的抗结核病筛药靶标。本申请拟针对目前尚未阐明功能的10种Mtb P450为研究对象，采用同源重组、基因关闭等手段对P450基因进行定点敲除，寻找和揭示Mtb存活或毒力必须的亚型，为新型筛药靶标的确立及其功能研究奠定基础。另外，本申请还拟以两种已知的Mtb存活必须 P450为靶，通过基因克隆表达、蛋白纯化手段获得两种酶的重组蛋白，经体外建立以P450为靶的高通量药物筛选模型，利用我国丰富的微生物资源，从我单位的微生物发酵产物和其他来源化合物库中筛选Mtb P450抑制剂，以期获得新型抗结核药物前体。

The war to combat the global threat from tuberculosis (TB) is challenged by the problem with the absence of new drugs and the emergence of drug-resistant strains in M. tuberculosis. One of the strategies to overcome the problem is to develop more novel targets for anti-tuberculosis screening. A series of studies in recent years revealed that P450s played very important roles in physiological events such as the metabolism and biosynthesis of lipid, the usage of cholesterol, and the electron transmission of redox in Mtb. A couple of the P450 members have been identified essential and indispensable for surviving and virulence of Mtb, and suitable for working as targets for anti-tuberculosis screening. To explore more effective targets from Mtb P450 family, this project will focus on 10 members of P450s whose functions remain unknown currently. By construction of targeted mycobacterial mutants by homologous recombination and gene switching,more P450 members being surviving/virulence essential are planned to be revealled in this project. In addition, two essential-known P450 will be used as targets for anti-tuberculosis HTS. Cloning and recombinant expressing will be carried out to obtained the purified P450s, and a well-designed HTS model will be established.A natural product and compound library which come from microbial ferment and other sources provided by our institute will be used for screening in this project.Any good hit will provided us a new candidate for future anti-tuberculosis development.