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

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-trans－ferase 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。

Abstract:

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，respec－tively（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 succin－imide 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.