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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 and screen the target gene of 2-C-methyl-D-erythritol-4-mycobacterium tuberculosis (MTB) phosphate cytidylyltransferase, Isp D), a novel anti-multidrug-resistant MTB drug with a novel mechanism of action was developed. Methods: High-throughput micro-organisms in vitro micro-level, rapid and rapid drug susceptibility test method screening positive compounds with MTB inhibitory growth; at the same time in Escherichia coli (Escherichia coli, E. coli) recombinant expression of MTB Isp D, established Enzyme-linked immunosorbent assay (ELISA), Isp D inhibitor screening model was established to screen Isp D inhibitors from positive compounds with MTB inhibitory activity. The mechanism of action of compounds with good inhibitory activity at the enzyme level was studied, The minimal inhibitory concentration (MIC) of H37Rv was evaluated. The antimicrobial spectrum was evaluated and its toxicity to Vero and Hep G2 was tested. RESULTS: A total of 159 positive compounds with MTB inhibitory activity were obtained from more than 40,000 compounds. Five inhibitors of Isp D were screened out, of which, IMB-4901 showed better inhibitory activity against Isp D (50 % inhibiting concentration, IC50 = 19.3 μg · m L-1) and anti-MTB activity (MIC = 1.6 μg · m L-1). Conclusion: A novel anti-MTB drug lead compound targeting Isp D was successfully obtained.