以苯丙氨酰-tRNA合成酶为靶点的新型抗结核药物高通量筛选模型的建立和应用

The establishment and application of a high throughput screening assay for inhibitors of Mycobacterium tuberculosis Phenylalanyl-tRNA synthetase

摘要:

【目的】建立结核分枝杆菌PheRS 抑制剂高通量模型, 并运用此模型筛选化合物和发酵液样品。【方法】克隆和表达结核分枝杆菌PheRS蛋白并优化其酶活测定方法, 在此基础上建立结核分枝杆菌PheRS 抑制剂高通量筛选模型, 并通过耻垢分枝杆菌作为检定菌对筛选到的样品进行抗菌活性测定及细胞毒性评价。【结果】运用此模型筛选了化合物样品11 600个, 发酵液样品5 200个, 筛选得到阳性化合物9个, 阳性发酵液37个。而后通过耻垢分枝杆菌作为检定菌的抗菌活性测定及细胞毒性评价后, 得到了6个发酵液阳性样品。【结论】建立的PheRS 抑制剂模型可成功用于化合物和微生物发酵液的高效筛选, 得到的6个发酵液阳性样品在酶水平和抗分枝杆菌方面均具有良好活性且毒性较低, 值得进一步研究。

Abstract:

[Objective] To establish a high-throughput drug screening model for screening inhibitors of Mycobacterium tuberculosis Phenylalanyl-tRNA synthetase (PheRS). [Methods] In this work, Mycobacterium tuberculosis pheRS gene was cloned and overexpressed in E. coli, and then the purified recombinant enzyme was used to establish an high-throughput drug screening model. After establishment and optimization of the screening assay, a primary screening was performed with compounds library and microbial fermentation collection in our institute, and their anti-bacterial activity and cytotoxicity were assessed further. [Results] 9 out of 11 600 compouds and 37 out of 5 200 microbial fermentation samples were identified as primary hits. [Conclusion] 6 microbial fermentation samples inhibited enzymatic activity of PheRS in vitro and growth of Mycobacterium smegmatis, and showed lower cytotoxicity.