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

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.