Characterization of phenoxyacetic acid compounds in inhibition of human glutathione-S-transferase Mu

To design and synthesize 4 phenoxyacetic acid compounds, characterize their inhibitory potencies on human glutathione-S-transferase Mu (glutathione-S-transferase Mu, GSTM) by different methods, and investigate the structure-activity relationship between phenoxyacetic acid compounds and GSTM.

Four phenoxyacetic acid compounds were designed and synthesized, and they are ethyl 2-(4-acryloylphenoxy) acetate (Ⅰa) and N, N’-(butane-1, 4-diyl)-bis-(4-acryloyl phenoxyacetic amide) (Ⅱa) containing α, β-unsaturated ketone structure, and ethyl 2-(4-propionylphenoxy) acetate (Ⅰb), N, N’-(butane-1,4-diyl)-bis-(4-propionyl phenoxyacetic amide) (Ⅱb) not containing α, β-unsaturated ketone structure. They were characterized by the half-maximal inhibitory concentration (IC₅₀) and apparent inhibition constant (K_i) using initial velocity method, and the dissociation constants (K_d) for GSTM utilizing fluorescence analysis.

Ⅰa and Ⅱa contained α, β-unsaturated ketone structure that undergoes electrophilic addition reaction with GSH. After incubation with enzyme and GSH, Ⅰa and Ⅱa generated products in situ, with a IC₅₀ value of 67.00±6.00 and 0.10±0.02 μmol/L for GSTM respectively (n=2). Ⅰb and Ⅱb without α, β -unsaturated ketone structures had no inhibitory potencies for GSTM. The K_i of Ⅰa against GSH was 19.9±1.6 μmol/L, and the K_i against CDNB was 9.1±0.7 μmol/L (n=3). The K_i of Ⅱa against GSH was 0.063±0.005 μmol/L, and against CDNB was 0.079±0.006 μmol/L (n=3). The K_i of Ⅱa was lower than that of Ⅰa (P<0.05). The K_d of Ⅰa, Ⅰb, Ⅱa, and Ⅱb measured by fluorescence analysis were 41.5±1.8, 38.9±1.7, 18.0±0.9, and 19.5±1.0 μmol/L, respectively (n=2). The K_d between Ⅰa and Ⅰb, and between Ⅱa and Ⅱb had no significant differences (P>0.05). The K_d of Ⅱa was lower than that of Ⅰa, and the K_d of Ⅱb was lower than that of Ⅰb (P<0.05).

α, β-unsaturated ketone structure is the essential functional group of phenoxyacetic acid compounds for inhibition on GSTM. Comparing with monovalent Ⅰa and Ⅰb, divalent Ⅱa and Ⅱb have higher affinity for GSTM. The product of Ⅱa has inhibitory potency significantly higher than that of Ⅰa.