药学学报, 2018, 53(6): 878-886
引用本文:
王霞, 朱宁屿, 姜威, 司书毅. 以莽草酸激酶为靶点的新型抗结核先导化合物的发现[J]. 药学学报, 2018, 53(6): 878-886.
WANG Xia, ZHU Ning-yu, JIANG Wei, SI Shu-yi. Identification of novel anti-tuberculosis lead compound targeting shikimate kinase[J]. Acta Pharmaceutica Sinica, 2018, 53(6): 878-886.

以莽草酸激酶为靶点的新型抗结核先导化合物的发现
王霞, 朱宁屿, 姜威, 司书毅
中国医学科学院、北京协和医学院医药生物技术研究所 国家新药 (微生物) 筛选实验室, 北京 100050
摘要:
莽草酸激酶是莽草酸途径中的关键蛋白,对结核分枝杆菌的生存至关重要。本研究首先建立了结核分枝杆菌莽草酸激酶(Mycobacterium tuberculosis shikimate kinase,MtSK)抑制剂筛选模型,然后与耻垢分枝杆菌表型筛选方法联用,对12万个化合物进行筛选,最终发现了1个对MtSK蛋白有抑制作用且同时抑制耻垢分枝杆菌生长的化合物—IMB-T5297[(E)-3-(3-(3-氯-5-甲氧基-4-(丙-2-炔-1-基氧基)苯基)丙烯酰基)-6-甲基-2H-吡喃-2,4(3H)-二酮]。体外酶促反应抑制实验表明,化合物IMB-T5297对MtSK的半数抑菌浓度(half maximal inhibitory concentration,IC50)为1.745 μg·mL-1。表面等离子共振(surface plasmon resonance,SPR)实验结果显示化合物IMB-T5297与MtSK蛋白的亲和力KD值为2.151×10-5 mol·L-1。通过计算机软件模拟化合物与蛋白的分子对接模型,初步分析了化合物IMB-T5297与MtSK的作用位点,并对其中预测的5个关键氨基酸位点进行了突变研究。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]实验表明,化合物IMB-T5297对哺乳动物细胞的毒性很低。体外抗结核活性实验表明,化合物IMB-T5297对结核分枝杆菌标准株H37Rv最低抑菌浓度(minimum inhibitory concentration,MIC)为49.723 μg·mL-1。综上所述,虽然化合物IMB-T5297的抗结核活性较弱,但对MtSK抑制作用较强,通过结构改造可能成为新的抗结核先导化合物。
关键词:    结核分枝杆菌      莽草酸激酶      药物筛选      抗结核药物     
Identification of novel anti-tuberculosis lead compound targeting shikimate kinase
WANG Xia, ZHU Ning-yu, JIANG Wei, SI Shu-yi
National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
Shikimate kinase is a key protein of the shikimic pathway, which is essential for the survival of Mycobacterium tuberculosis. In this study, a screening assay for Mycobacterium tuberculosis shikimate ki-nase (MtSK) inhibitor was developed. A 120 000-compound library was screened by the enzyme assay and the phenotype screening using Mycobacterium smegmatis. A hit compound named IMB-T5297[(E)-3-(3-(3- chloro-5-methoxy-4-(prop-2-yn-1-yloxy)phenyl)acryloyl)-6-methyl-2H-pyran-2,4(3H)-dione] was identified to be a selective inhibitor of MtSK with a half maximal inhibitory concentration (IC50) value of 1.745 μg·mL-1, which also showed antibacterial activity. The interaction between compound and protein was analyzed by surface plasmon resonance (SPR) experiment, which showed the KD value was 2.151×10-5 mol·L-1. The binding model of MtSK and compound was simulated by the computer program. Five key amino acids in the binding pocket were indispensable site-directed mutated to verify the model. IMB-T5297 inhibited Mycobacterium tuberculosis H37Rv with a minimum inhibitory concentration (MIC) value of 49.723 μg·mL-1 and displayed low cytotoxicity to mammalian cells. In this study, IMB-T5297 was identified as a selective inhibitor of MtSK enzyme with anti-tuberculosis activity. With additional structural modification, the compound has a potential to become a novel anti-tuberculosis compound.
Key words:    Mycobacterium tuberculosis    shikimate kinase    drug screening    anti-tuberculosis drug   
收稿日期: 2018-03-31
DOI: 10.16438/j.0513-4870.2018-0284
基金项目: 国家自然科学基金资助项目(81573474);北京协和医学院小规模特色办学专项青年教师培养项目(2015zlgc0745).
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