基于反式翻译过程筛选抗结核活性化合物的作用机制及候选靶标确认

The global control and management of tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a major health threat. The global control and management of tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a major health threat. The success of Mtb as a pathogen is partly due to its ability to persist in host microenvironments. Control of TB is faced with the formidable challenge of worsening scenarios of drug-resistance and persistence of Mtb. Identification of new mechanisms of drugs is very important to control transmission of Mtb and reactivation from latent tuberculosis. However, lack of novel and validated drug targets limits the progress of drug research and development. Pharmacological validation of a potential target is an important prerequisite for drug discovery, and few such targets are known in Mtb..Pyrazinamide (PZA) is a unique first-line drug used for the treatment of TB. It plays a key role in killing non-replicating persisters that other TB drugs fail to kill, which makes it an essential drug for inclusion in any drug combinations for treating drug-susceptible and drug resistant TB such as multidrug-resistant TB. RpsA (ribosomal protein S1), which is involved in process of trans-translation, was identified as a target of PZA based on its binding activity to pyrazinoic acid (POA), the active form of PZA in our previous research (Science, 2011). In addition, we reported the crystal structures of the C-terminal domain of RpsA of Mtb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance (Mol. Micro.2015). The pharmacological validation of PZA target proposed that the trans-translation machinery, in particular, the RpsA protein maybe an excellent target for the development of novel antituberculosis. .On the basis of structural data, screening was performed in a search for novel binding molecules of RpsA, and several compounds with antituberculosis activity were screened in vitro. In this project, the activity of compounds against active Mtb and persisters will be further evaluated. In order to validate the reliability of RpsA and trans-translation process as the novel targets against TB, a series experiments on interaction between active compounds and RpsA will be performed. Moreover, effects of compounds on tmRNA binding activities of RpsA proteins and on trans-translation progress will be tested. Our studies may uncover RpsA and trans-translation process as novel targets for chemotherapeutic intervention. Screening of inhibitors of trans-translation progress may be an attractive strategy for development of novel antibiotics against TB.

结核杆菌（MTB）是引起结核病（TB）的罪魁祸首，杀灭耐药和潜伏MTB是控制结核病的关键，开发新型药物对于控制耐药结核传播和潜伏菌复发至关重要。药物研发瓶颈在于靶点的不确定性，而靶点的药理学验证才是确证靶点真实性的首要必备条件。丙嗪酰胺（PZA）是一个独特抗结核药物，具有抑制潜伏结核杆菌活性并是结核病（敏感和耐药）治疗中唯一不可替代的药物。反式翻译过程关键蛋白RpsA是我们前期发现的PZA作用靶点（Science，2011），且通过结构解析蛋白与药物分子作用模式而明确的新型抗结核潜在靶点（Mol. Micro.2015）。在此靶点基础上，筛选到具有抗结核杆菌活性的化合物，本项目拟继续通过活性化合物对反式翻译过程及RpsA影响及作用机制的深入研究来揭示RpsA及反式翻译过程作为新型抗结核靶点的真实可靠性。研究成果为发现新型抗结核药物靶标提供基础，对开发源头创新的抗结核杆菌药物有着重要意义。

结核杆菌是引起结核病的罪魁祸首，药物化疗依然是控制结核的重要手段，开发新型药物对于控制耐药结核传播和潜伏菌复发至关重要。本项目在前期基于反式翻译过程关键蛋白RpsA筛选到的化合物基础上，进一步通过体外和细胞内的抗结核评价并筛选出一个具有良好抗结核活性的先导化合物，并初步揭示其抗结核的作用机制。项目通过建立可视化启动反式翻译途径工程菌株及DARTs技术研究发现活性化合物不能直接与RpsA结合，可与ClpX和HisD蛋白结合，但进一步发现该化合物并不能直接抑制ClpX及HisD蛋白的酶活性。为进一步探索活性化合物抗结核的作用机制，将化合物作用于结核杆菌后进行转录组学分析，发现FAS-II脂肪酸合成通路rv2243(fabD), rv2244(acpM), rv2245(kasA), rv2246(kasB), rv2247(accD6)基因显著上调。同时所筛选的化合物耐药结核杆菌全基因组测序发现，四株高耐药菌株均存在hadA基因存在非同义突变，hadC基因存在单碱基缺失造成的移码突变。hadA和hadC基因是FAS-II脂肪酸合成通路中的脱水酶，HadA和HadC需要与HadB形成复合物后，在结核杆菌长链脂肪酸合成中发挥重要作用。进一步将这三个基因分别在H37Ra中单独过表达或同时两个或三个共同过表达，MIC测定发现除过表达HadA不会造成化合物耐受外，其他过表达菌株均产生不同程度的化合物耐受，特别是过表达HadBC和HadABC会发生高度耐药。DARTS及ITC实验结果表明化合物可以与HadAB及HadBC结合。单向和双向TLC分析发现化合物作用导致分枝杆菌TDM和TMM的合成受损。综上研究表明化合物通过与HadA，HadC结合并影响HadAB，HadBC复合物的形成，进而影响FASII通路导致长链脂肪酸的合成受阻，进而达到抑制结核杆菌的目的，研究为发现新型抗结核药物和药物靶点提供重要价值。