| Glutathione-S-transferase (glutathione-S-transferases, GST) as a group protein with a variety of biological functions widely exist in mammals, It features as a dimer of two composed of identical subunits; each sub-base has an activity center, composed of glutathione binding site (G-site) and adjacent sparse underwater objects binding sites (H-site). GST inhibitors may enhance the antitumor efficacy of cytotoxic drugs or directly inhibit tumor growth.Therefore, the design and screening of GST inhibitors become the focus of the study.This paper formulated the enzyme kinetics method for the determination inhibition capacity of GST inhibitors, screened two series of GST inhibitors that were designed and synthesized.1Preparation and characterization of liver acidic GST isozymeThe acidic GST isozyme was purified from the porcine liver via anion-exchange chromatography and affinity chromatography. Specific activity of GST was increased by more than146times with overall activity yield of about30%. GST followed random bi-substrate kinetics and had Km of42μmol/L for GSH, and Km of0.86mmol/L for CDNB.2Enzyme kinetics method of for the determination of inhibition ability on Glutathion-S-transferaseThe reaction of glutathione (glutathione,GSH) and1-chloro-2,4-dinitr-obenzene (1,-chloro-2,4-dinitrobenzene,CDNB) gave S-(2,4-dinitrobenzyl)-glutathione(GS-DNB) as a candidate inhibitor. Michaelis-Menten constant (Km) and maximal reaction rate (Vm) were estimated to determine the inhibition constant (Ki) of GS-DNB. The competitive Ki of GS-DNB was (21±1) umol/L (n=2) against CDNB, and (17±1) umol/L (n=2) against GSH. GS-DNB is an effective competitive inhibitor of GST; the estimation of Ki from responses of Km and Vm to inhibitor concentrations can be a conventional method to screen GST inhibitors.3Design and screening of Glutathione-S-transferase inhibitors Referring to lipid-water partition coefficient (LogP), two series of GST inhibitors were designed:the first series are the conjugates of hydrophobic compounds like P-aminobenzoic acid with GSH; the second are the symmetrical diamides of ethacrvnic acid (EA),4-butyl benzoic acid,3,5-dimethyl-benzoic acid, dansyl acid and other carboxylic acid as hydrophobic binding groups, linked with1,3-propanediamine,1,5-pentane-diamine,lysine and1,4-diaminobutane as a spacers.By enzyme kinetics method to determination inhibition constant (Ki), N,N’-bis-ethacrynyl-1,4-butyldiamine and N,N’-bis-(4-(n-butyl)-benzoyl)-1,4-butyldiamine displayed strong competitive inhibition on this GST against GSH with Ki of38nmol/L and0.28umol/L.which were also stronger than ethacrvnic acid and4-butyl benzoic acid, respectively. But they all bound to just one active site of GST in theory due to the mlecular sizes. Thus, symmetrical structures of low-affinity inhibitors linked via short linear spacers had promise to be high-affinity inhibitors against just one H-site of GST,and new symmetrical structures against two H-sites of GST are preferable as much stronger inhibitors.Taken together, the enzyme kinetics mehod to screen GST inhibitor was estabilished, newly designed GST inhibitors showed high-affinity inhibitions.This work layed the foundation for the discovery of new GST inhibitors as antitumor chemotherapytic sensitizers. |
