Design, Synthesis And Characterization Of Divalent Pro-inhibitors Of Glutathione-S-transferase Isozyme M

Glutathione-S-transferases (GST; EC2.5.1.18) are antidotal enzymeswhich catalyze the conjugation of glutathione (GSH) to various kinds ofelectrophilic substrates.There are several classes of isoenzymes (e.g.，μ，ω，π，θ，ζ).hGSTπ (hGSTP) is mainlyinduced incancer cells and its actionsresulted in drug resistance. Inhibitors of hGSTP are agents to overcome drugresistance of chemotherapy. However, it was found that hGSTM can beinduced in some drug-resistant tumor cells. As a consequence, rationaldesign of GSTM-selective pro-inhibitors is promising to yield leads ofpotent drugs for sensitizing drug-resistant cancers.In this study, a series of hGSTM specific pro-inhibitors were designedbased on differences in structures of hGSTM, hGSTA hGSTP. hGSTMdimer has the shortest and most open cleft that can serve as an additionalbinding domain to enhance the selectivity of designed inhibitor. Somesuitable linear linkers double binded with monovalent pro-inhibitors yielddivalent pro-inhibitors that can produce conjugations of GSH under theaction of GST with much stronger inhibitions. Pro-inhibitors have thefreedom to across cell membrane due to hydrophobicity.Ethacrynic acid (EAA) is a classical monovalent inhibitor of GSTwhose inhibition constant (Ki) is at micromolar levels. The linkage ofethanediamine, butanedimine, hexanedimine, etc, to two EAA and two 4-acryloyl phenyl unit, respectively, to yield two series of divalentpro-inhibitors; their glutathione conjugates have apparent inhibitionconstant to hGSTM below30%of those to hGSTP or hGSTA；the GSHconjugates of divalent pro-inhibitors were slow tight-binding selectiveinhibitors of hGSTM.GST has two substrates and its kinetic mechanics is complicated, the Kifor two substrates are different. Hence, two methods were developed toassay the dissociation constants of inhibitors. The first was enzymicdynamic method to measure Kiof inhibitors through nonlinear fitting toresponse curves of enzyme activity to inhibitor concentrations. The secondwas fluorometric method to measure Kdof inhibitors through nonlinearfitting to response curves of fluorometric intensity of GST at340nm toinhibitor concentration. Two methods gave the consisitent results to supporttheir reliability.In conclusion, pro-inhibitors specific for hGSTM were designed andsynthesized, their products acted as slow tight-binding inhibitors with thehighest affinity so far. Two new methods gave their consistent affinitieslower than10nM. These achievements may provide assistance for furtherstudy on agents for sensitizing tumor drug resistance.