抗耐药结核病新药吡法齐明通过醌氧化还原酶介导发挥作用的机制研究  

Study of the Mechanism of the New Antidrug-resistant Tuberculosis Drug Pyrifazimine Mediated by Quinone Oxidoreductase

作　　者：张蕾 李媛媛 陈曦[1] 刘海婷 徐建[1] 王宁 丁杨明 陆宇[1] Zhang Lei;Li Yuanyuan;Chen Xi;Liu Haiting;Xu Jian;Wang Ning;Ding Yangming;Lu Yu(Beijing Key Laboratory of Drug Resistance Tuberculosis Research,Department of Pharmacology,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing Chest Hospital,Capital Medical University,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京胸科医院耐药结核病研究北京市重点实验室北京市结核病胸部肿瘤研究所药物研究室,101149

出　　处：《结核病与胸部肿瘤》2021年第4期284-290,共7页Tuberculosis and Thoracic Tumor

基　　金：北京市自然科学基金(7212150);“十三五”国家科技重大专项(2019ZX09721001-007-003);北京市医院管理中心临床医学发展专项(ZYLX202123)。

摘　　要：目的对批法齐明(pyrifazimine,TBI-166)通过醌氧化还原酶(quinone oxidoreductase,QOR)介导累积活性氧(reactive oxygen species,ROS)发挥抗结核作用的机制进行探索。方法表达、纯化MtbQOR蛋白,设置空白对照组及加TBI-166(0.03、0.3、3μg/ml)组,酶标仪监测波长340 nm处吸光度(A_(340nm))下NADPH的下降程度(△A);应用辅酶ⅡNADP(H)含量检测试剂盒检测不同浓度TBI-166作用H37Rv菌体后NADPH/NADP+的比值;应用细菌活性氧检测试剂盒检测结核分枝杆菌H37Rv标准株和临床分离株菌体内ROS荧光值。结果空白对照组△A(0.316±0.032)较0.03、0.3、3μg/ml TBI-166组(0.506±0.107、0.531±0.054、0.801±0.079)明显降低,差异有统计学意义(t=-3.799,P<0.05;t=-7.634,P<0.01;t=-12.683,P<0.01)。在48h内,加TBI-166组NADPH/NADP^(+)比值(0.847±0.229)低于空白对照组比值(1.728±0.384)。H37Rv菌株中,TBI-166刺激组ROS荧光值(23072±2584)明显高于空白对照组(8282±448),差异有统计学意义(F=33.334,P<O.O1)。在菌株号为12897菌株中,TBI-166刺激组ROS荧光值(18369±3118)明显高于空白对照组(12268±2735),差异有统计学意义(F=5.026,P<0.05);在菌株号为15171菌株中,3μg/ml TBI-166刺激组ROS荧光值(17572±249)明显高于空白对照组(11109±343),差异有统计学意义(F=122.601,P<0.01);在菌株号为11492及11195菌株中,TBI-166刺激组与空白对照组ROS荧光值差异无统计学意义(F=161.440,P>0.05;F=44.788,0.05).结论TBI-166可以通过增加MtbQOR介导的氧化还原反应,降低NADPH/NADP^(+)的比值,刺激菌体累积ROS发挥抗结核作用。Objective To study the mechanism of pyrifazimine(TBI-166)accumulating reactive oxygen species(ROS)to kill Mycobacterium tuberculosis(MTB)through quinone oxidoReductase(QOR).Methods MtbQOR protein was expressed and purified.The decrease of NADPH(△A)at A340 nni in blank control group and TBI-166(0.03>0.3>3μg/ml)groups were detected by microplate reader;NADPH/NADP+ratio of H37Rv after exposure to TBI-166 of different concentrations were detected by coenzyme II NADP(H)detection kit;ROS fluorescence value of H37Rv strains and clinical collected strains were tested by bacterial ROS detection kit.Results The△A of control group(0.316±0.032)was lower than that of 0.03,0.3,3μg/ml TBI 166 groups(0.506±0.107,0.531±0.054,0.801±0.079)(t=-3.799,P<0.05;t=-7.634,P<0.01;t=-12.683,P<0.01).Within 48 hours,the NADPH/NADP^(+)ratio of TBI-166 groups(0.847±0.229)was lower than that of the control group(1.728±0.384).ROS fluorescence value of H37Rv strains in TBI-166 groups(23072±2584)was higher than that of the control group(8282±448)(F=33.334,P<0.01).ROS fluorescence value of the strain number 12897 in TBI-166 groups(18369±3118)was higher than that of the control group(12268±2735)(F=5.026,P<0.05).ROS fluorescence value of the strain number 15171 in 3μg/ml TBI-166 group(17572±249)was higher than that of the control group(11109±343)(F=122.601,P<0.01).There was no significant difference detected in ROS fluorescence value between TBI-166 groups and blank control group in the strains numbered 11492 and 11195(F=161.440,P>0.05;F=44.788,P>0.05).Conclusion TBI-166 could enhance redox reaction mediated by MtbQOR,decrease the ratio of NADPH/NADP^(+)and accumulate intra-bacterial ROS to kill MTB.

关 键 词：分枝杆菌 结核 药理作用分子机制 醌氧化还原酶 吡法齐明 

分 类 号：R73[医药卫生—肿瘤]