以肽脱甲酰基酶为靶点的抗结核药物高通量筛选模型的建立和应用

目的建立以肽脱甲酰基酶(PDF)为靶点的抗结核药物高通量筛选模型,应用该模型筛选得到活性微生物发酵液粗提物样品。方法以结核分枝杆菌H37Rv基因组为模板,扩增肽脱甲酰基酶的基因片段def,构建表达载体pET-28a-def,表达并纯化结核分枝杆菌PDF酶;基于PDF水解三肽底物for-Met-Ala-Ser释放出游离NH2,而游离NH2可与荧光胺反应产生荧光的原理,利用测定所产生荧光值的方法,建立高通量药物筛选模型;使用该模型对12400个微生物发酵液粗提物样品进行筛选,同时以耻垢分枝杆菌为检定菌,平板纸片法检测样品的抗菌活性,并检测所得阳性样品的细胞毒性。结果成功构建了表达载体pET-28a-def;所建立的模型稳定可行,可用于以肽脱甲酰基酶为靶点的抗结核药物的高通量筛选;用该模型对12400个微生物发酵液粗提物样品进行筛选,最终得到8个对肽脱甲酰基酶抑制活性和抗耻垢分枝杆菌活性均较好的阳性样品,阳性率0.06%;其中5个样品的细胞毒性较小。结论建立了灵敏度好、稳定性高的结核分枝杆菌肽脱甲酰基酶抑制剂高通量药物筛选模型,应用该模型所得到的阳性样品具有进一步深入研究的意义。

The establishment and application of high throughput screening model targeting Mycobacterium tuberculosis peptide deformylase

Objective To establish and validate a high throughput model for screening of Mycobacterium tuberculosis peptide deformylase (PDF) inhibitors as potential antituberculosis drugs, and to perform preliminary screening with microbial fermentation extracts. Methods The H37Rv PDF coding gene def was amplified with PCR and cloned into the expression vector pET-28a. The recombinant PDF was over-expressed and purified. A high throughput model was established based on the detection of fluorescence intensity, which is caused by the reaction of fluorescamine and free NH2 released by PDF from substrate for-Met-Ala-Ser. Using the assay, 12 400 microbial fermentation extracts were screened, and positive samples’ antibacterial activity to Mycobacterium smegmatis mc2 155 were tested using K-B method. Finally, cytotoxicity of positive samples was assessed. Results The expression vector pET-28a-def was constructed. The established model was feasible and stable for drug screening. 12 400 microbial fermentation extracts were screened and 8 positive samples were obtained, showing a 0.06% positive rate. Five of them had relatively low cytotoxicity. Conclusion In this work, we developed a sensitive and reproducible assay for screening of PDF inhibitors. The positive samples are worth investigating in the future.