两种依他尼酸衍生物对人类谷胱甘肽-S-转移酶Mu的抑制动力学研究  

A study of the inhibition kinetics of human glutathione-S-transferase Mu by two ethacrynic acid derivatives

作　　者：许榜田 赵语 龙友琦 白书连 杨晓兰[2] Xu Bangtian;Zhao Yu;Long Youqi;Bai Shulian;Yang Xiaolan(I.Pharmaceutical Department,University-Town Hospital of Chongqing Medical University;Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry,College of Laboratory Medicine,Chongqing Medical University)

机构地区：[1]重庆医科大学附属大学城医院药学部,重庆401331 [2]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《重庆医科大学学报》2020年第8期1144-1150,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:31570862)。

摘　　要：目的:考察所设计依他尼酰乙胺、双依他尼酰丁二胺及对应产物对人类谷胱甘肽-S-转移酶Mu(glutathione-S-transferase Mu,GSTM)的抑制动力学。方法:重组表达GSTM,以2,4-二硝基氯苯(1-chloro-2,4-dinitrobenzene,CDNB)和还原型谷胱甘肽(glutathione,GSH)为底物,跟踪酶反应过程的紫外吸收变化,测定酶活性。比较GSTM存在下依他尼酰乙胺、双依他尼酰丁二胺与GSH孵育前后对酶的半抑制浓度(IC50)、抑制类型。结果:依他尼酰乙胺、双依他尼酰丁二胺对GSTM的IC50分别为(44.0±2.2)μmol/L、(0.090±0.012)μmol/L(n=3),两者相对GSH是反竞争性抑制剂、相对CDNB是竞争性抑制剂。与酶和GSH孵育原位生成产物后,对GSTM的IC50分别为(8.9±0.1)μmol/L和(0.006±0.001)μmol/L(n=3);依他尼酰乙胺与GSH孵育后相对GSH是竞争性抑制剂、相对CDNB是混合型抑制剂;双依他尼酰丁二胺与GSH孵育后相对GSH和CDNB均为混合型抑制剂。两种依他尼酸衍生物与GSH孵育后产物IC50明显降低,双依他尼酰丁二胺及其GSH孵育产物IC50较依他尼酰乙胺及其产物均明显降低。结论:依他尼酰乙胺、双依他尼酰丁二胺是潜抑制剂,其产物对GSTM的抑制作用更强,抑制类型发生明显改变;双依他尼酰丁二胺及其产物较依他尼酰乙胺抑制作用更强。Objective:To investigate the inhibition kinetics of human glutathione-S-transferase Mu(GSTM) by ethacrynic ethylamide,bis-ethacrynic succinimide,and their corresponding products. Methods:GSTM was recombinantly expressed,and enzyme activity was determined by tracking changes in ultraviolet absorption using 1-chloro-2,4-dinitrobenzene(CDNB) and glutathione(GSH) as substrates. Half-maximal inhibitory concentrations(IC50) and inhibition types were compared between ethacrynic ethylamide,bis-ethacrynic succinimide,and their corresponding products before and after incubation with GSH in the presence of GSTM. Results:IC50 values of ethacrynic ethylamide and bis-ethacrynic succinimide against GSTM were(44.0±2.2) μmol/L and(0.090±0.012) μmol/L,respectively. They were uncompetitive inhibitors against GSH but competitive inhibitors against CDNB. After incubation with the enzyme and GSH,the IC50 values of these two agents against GSTM were(8.9±0.1) μmol/L and(0.006±0.001) μmol/L,respectively(n =3). Ethacrynic ethylamide turned to be a competitive inhibitor against GSH but a mixed competitive inhibitor against CDNB after incubation with GSH,while bis-ethacrynic succinimide was a mixed competitive inhibitor against both GSH and CDNB after incubation. Both ethacrynic acid derivatives had significantly decreased IC50 values after incubation with GSH.Furthermore,bis-etha-crynic succinimide and its GSH incubation products showed a reduction in IC50 value compared with ethacrynic ethylamide and its products. Conclusion:Ethacrynic ethylamide and bis-ethacrynic succinimide are pro-inhibitors,and their products have significant reduction in the IC50 values against GSTM after incubation with GSH. Meanwhile,their inhibitory types also change significantly. Bis-ethacrynic succinimide along with its products have significantly enhanced inhibition potencies compared with ethacrynic ethylamide.

关 键 词：人类谷胱甘肽-S-转移酶Mu 依他尼酸衍生物 潜抑制剂 抑制动力学 

分 类 号：Q555.8[生物学—生物化学]