摘要
目的:筛选靶向结核分枝杆菌(Mycobacterium tuberculosis,MTB)2-C-甲基-D-赤藓糖醇-4-磷酸-胞嘧啶转移酶(2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase,Isp D)的新型抗结核化合物,研制具有全新作用机制的新型抗多药耐药MTB药物。方法:应用高通量的微生物体外微量、直观快速的药敏试验方法,筛选得到具有抑制MTB生长的阳性化合物;同时在大肠杆菌(Escherichia coli,E.coli)中重组表达MTB的Isp D,建立酶活测定方法,构建Isp D酶抑制剂筛选模型,从具有抑制MTB生长的阳性化合物中筛选Isp D抑制剂;对在酶水平上具有良好抑制活性的化合物进行作用机制研究,确定其对MTB(H37Rv)的最低抑菌浓度(minimal inhibitory concentration,MIC),评价其抗菌谱,测定其对非洲绿猴肾细胞(Vero)和人肝癌细胞(Hep G2)的毒性。结果:从4万多个化合物中初步得到了159个具有抑制MTB生长的阳性化合物,从中筛选得到了5个Isp D的抑制剂,其中的IMB-4901显示出较好的Isp D抑制活性(50%inhibiting concentration,IC50=19.3μg·m L-1)和抗MTB活性(MIC=1.6μg·m L-1)。结论:成功获得了以Isp D为靶点的新型抗MTB药物先导化合物。
Objective： To screen novel antitubercular compounds targeting 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase（ Isp D） of Mycobacterium tuberculosis（ MTB）,and develop new anti-MDR MTB drugs with novel mechanism of action. Methods： Based on the high through-put in vitro microbial micro-assay with celerity and sensitivity,positive compounds inhibiting the growth of MTB were screened out. At the same time,recombinant MTB Isp D was expressed in E. coli. A method of activity assay for Isp D was established. Screening model for Isp D inhibitors was constructed. Inhibitors of Isp D were screened from positive compounds inhibiting the growth of MTB. Studies on the mechanism of compounds with strong inhibitory activity at enzymatic level were performed.The minimal inhibitory concentration（ MIC） of compounds for MTB（ H37Rv） were determined. The antibacterial spectrum was tested. The cytotoxicity on Vero and Hep G2 were also measured. Results： One hundred and fiftynine hits with antitubercular activity were found from about 40 000 candidates. Five Isp D inhibitors were screened out from the 159 hits. Compound IMB-4901 showed preferable enzymatic inhibitory activity（ IC50= 19. 3μg·m L- 1） and antitubercular activity（ MIC = 1. 6 μg·m L- 1）. Conclusion： A novel antitubercular lead compound targeting Isp D has been successfully found.
出处
《中国新药杂志》
CAS
CSCD
北大核心
2015年第17期1947-1953,共7页
Chinese Journal of New Drugs
基金
国家"重大新药创制"科技重大专项(2015ZX09304006-009)
国家自然科学基金(81202567)
