Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study it has been shown that ethacrynic acid can inhibit glutathione S-transferase (GST) of the pi-class irreversibly. [14C]Ethacrynic acid, 0.8 nmol/nmol human P1-1 and 0.8 nmol/nmol rat GST 7-7 could be incorporated, resulting in 65-93% inhibition of the activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Isoenzymes of the alpha- and mu-class also bound [14C]ethacrynic acid, however without loss of catalytic activity. Incorporation ranged from 0.3 to 0.6 and 0.2 nmol/nmol enzyme for the mu- and alpha-class GST isoenzymes, respectively. For all isoenzymes, incorporation of [14C]ethacrynic acid could be prevented by preincubation with tetrachloro-1,4-benzoquinone, suggesting, that a cysteine residue is the target site. Protection of GST P1-1 against inhibition by ethacrynic acid by the substrate analog S-hexylglutathione, indicates an active site-directed modification. The monobromo and dibromo dihydro derivatives of ethacrynic acid were synthesized in an effort to produce more reactive compounds. The monobromo derivative did not exhibit enhanced irreversible inhibitory capacity. However, the dibromo dihydro derivative inhibited both human and rat GST isoenzymes of the pi-class very efficiently, resulting in 90-96% inhibition of the activity towards CDNB. Interestingly, this compound is also a powerful irreversible inhibitor of the mu-class GST isoenzymes, resulting in 52-70% inhibition. The two bromine atoms only marginally affect the strong (reversible) competitive inhibitory capacity of ethacrynic acid, with IC50 (microM) of 0.4-0.6 and 4.6-10 for the mu- and pi-class GST isoenzymes, respectively.
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PMID:Isoenzyme selective irreversible inhibition of rat and human glutathione S-transferases by ethacrynic acid and two brominated derivatives. 844 64

Glutathione (GSH) and glutathione-related enzyme systems in astrocytes play an important role in cellular defense against oxidative stress in the nervous system. The present study was designed to characterize the cellular responses of cultured astrocytes to chemically-induced perturbations of mitochondrial and cytosolic GSH homeostasis. Treatment of astrocytes in culture with ethacrynic acid (EA), a mitochondrion-penetrating thiol reagent, induced rapid and extensive depletion of both cytosolic and mitochondrial pools of GSH. Concomitant with the effects of EA on cellular GSH were significant and concentration-dependent increases in intracellular generation of reactive oxygen species (ROS) as indicated by the oxidation of preloaded 2',7'-dichlorofluorescein diacetate. Significant elevation of intracellular ROS occurred by 15 min following exposure to 100 microM EA and reached peak levels by 30 min which were approximately 7-fold higher than corresponding control levels. Ethacrynic acid-induced GSH depletion and intracellular ROS elevation was followed by marked decreases in glutathione reductase (GR) activity in mitochondria, and to a lesser extent, in cytosolic fractions of cultured astrocytes. This inhibitory effect was time- and concentration-dependent, and other GSH-related enzymes, glutathione peroxidase and glutathione S-transferase, were not or only slightly affected. Kinetic studies showed that EA markedly diminished V(max) values of both mitochondrial and cytosolic GR without affecting K(m), suggesting noncompetitive inhibition of this thiol-dependent enzyme. Another thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase was also markedly inhibited by EA in a time-dependent fashion. Subsequent decline of mitochondrial transmembrane potential (rhodamine 123 uptake) and cellular ATP production following EA treatment occurred prior to the onset of loss of cell viability as indicated by lactate dehydrogenase leakage. These results suggest that the loss of mitochondrial GSH may render the astrocytes unable to combat the pathological sequelae of endogenous oxidative stress, leading to perturbations of thiol-dependent enzyme activities, mitochondrial function and energy metabolism.
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PMID:Cellular responses of cultured cerebellar astrocytes to ethacrynic acid-induced perturbation of subcellular glutathione homeostasis. 868 Aug 62

The potent diuretic drug ethacrynic acid has been tested in clinical trials as an adjuvant in chemotherapy. Its target is the detoxifying enzyme glutathione transferase which is often found overexpressed in cancer tissues. We have solved the crystal structures of human pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Ethacrynic acid is found to bind in a nonproductive mode to one of the ligand binding sites of the enzyme (the H site) while the glutathione binding site (G site) is occupied by solvent molecules. There are no structural rearrangements of the G site in the absence of ligand. The structure indicates that bound glutathione is required for ethacrynic acid to dock into the H site in a productive binding mode. The binding of the ethacrynic acid-glutathione conjugate shows that the contacts of the glutathione moiety with the protein are identical to those observed in crystal structures of the enzyme with other glutathione-based substrates and inhibitors. The ethacrynic acid moiety of the conjugate binds in the H site in a fashion that has not been observed in crystal structures of other glutathione-based inhibitor complexes. The crystal structures implicate Tyr 108 as an electrophilic participant in the Michael addition of glutathione to ethacrynic acid.
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PMID:The three-dimensional structure of the human Pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. 901 73

Ethacrynic acid (EA), a diuretic drug, is known to interact with glutathione transferases in the presence of reduced glutathione (GSH) to yield an EA-SG conjugate. Here we present evidence for a new mechanism for the formation of EA-SG conjugate by a soybean lipoxygenase (SLO)-mediated reaction involving oxidation of GSH to a GS.. Similar to the glutathione transferase-mediated reaction, EA-SG conjugate generated by SLO exhibited an absorbance maximum at 270 nm. The conjugate formation was dependent on the concentration of linoleic acid, EA, GSH, and SLO. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.4 mM linoleic acid, 1 mM GSH, 50 nM SLO, and 0.2 mM EA at pH 9.0. Classical inhibitors of lipoxygenase, e.g., nordihydroguaiaretic acid, gossypol, and 5,8,11-eicosatriynoic acid, significantly inhibited EA-SG conjugation. The SLO-generated EA-SG was isolated as a single peak by HPLC. Quantitation of EA-SG by HPLC-coupled radiometry using [3H]GSH yielded a rate of 16.5 mumol/min/mg SLO protein. This rate is up to 1650-fold greater than that reported for different purified isozymes of mammalian glutathione transferase. The structure of EA-SG isolated from HPLC column was confirmed by matrix-assisted laser desorption mass spectroscopy. These results suggest that lipoxygenase, which is primarily known for xenobiotic oxidation, may represent yet another important pathway for GSH conjugate formation that could lead to detoxification of certain chemicals.
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PMID:A novel mechanism of glutathione conjugate formation by lipoxygenase: a study with ethacrynic acid. 907 6

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.
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PMID:Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells. 1150 Jan 36

Inorganic arsenicals are important environmental toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethyarsinic acid (DMA). Recent evidence indicates DMA is a complete carcinogen in rodents while evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMA compared to that of the trivalent inorganic arsenical, sodium arsenite, using a rat liver epithelial cell line (TRL 1215). Arsenite was very cytotoxic in these cells (LC(50) = 35 microM after 48 h of exposure). With arsenite exposure, most dead cells showed histological and biochemical evidence of necrosis. Arsenite cytotoxicity increased markedly when cellular GSH was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO). In contrast, DMA was nearly 3 orders of magnitude less cytotoxic (LC(50) = 1.5 mM) although evidence showed the predominating form of death was apoptosis. Surprisingly, GSH depletion actually decreased DMA-induced apoptosis. A glutathione scavenger, diethyl maleate (DEM), and a glutathione reductase inhibitor, carmustine, also prevented DMA-induced apoptosis. These data indicate that DMA requires intracellular GSH to induce apoptosis. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyzes GSH-substrate conjugation, acivicin, an inhibitor of gamma-glutamyltranspeptidase (GGT) which catalyzes the initial breakdown of GSH-substrate conjugates, and aminooxyacetic acid (AOAA), an inhibitor of beta-lyase which catalyzes the final breakdown of GSH-substrate conjugates, all were effective in suppressing DMA-induced apoptosis. These findings indicate that DMA likely is conjugated in some form with GSH, and that it is this conjugate that induces apoptosis during subsequent metabolic reactions.
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PMID:A major human arsenic metabolite, dimethylarsinic acid, requires reduced glutathione to induce apoptosis. 1201 83

We examined the effect of nipradilol on contraction of the posterior ciliary artery induced by high potassium or norepinephrine and on cyclic GMP (cGMP) levels in the posterior ciliary artery of dogs. Nipradilol caused dose-dependent relaxation of KCl-and norepinephrine-induced contractions of posterior ciliary artery. The relaxant effect of nipradilol on norepinephrine-contracted ciliary artery was significantly greater than that on KCl-contracted ciliary artery. In KCl-contracted ciliary artery, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 10(-4) M) did not alter the relaxant effect of nipradilol, whereas 1H-1,2,4-oxadiazolo-4,3-a-quinoxalin-1-one (ODQ, 10(-6) M) significantly inhibited this effect. Ethacrynic acid at 10(-5) M, sulfasalazine at 10(-4) M and S-decylglutathione at 10(-4) M (glutathione S-transferase inhibitors) did not inhibit the relaxant effect of nipradilol. In addition, nipradilol produced dose-dependent increases in cGMP levels in the canine posterior ciliary artery. These findings indicate that nipradilol-induced vasorelaxation in the canine posterior ciliary artery occurs via stimulation of the guanylyl cyclase-cGMP pathway.
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PMID:Nipradilol induces vasodilation of canine isolated posterior ciliary artery via stimulation of the guanylyl cyclase-cGMP pathway. 1209 33

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAs(V)) and dimethylarsinic acid (DMAs(V)). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAs(V). We studied the molecular mechanisms of in vitro cytolethality of MMAs(V) using a rat liver epithelial cell line (TRL 1215). MMAs(V) was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, l-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH-substrate conjugation, also enhanced the cytolethality of MMAs(V), but aminooxyacetic acid (AOAA), an inhibitor of beta-lyase that catalyses the final breakdown of GSH-substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAs(V) in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAs(V), although cellular GSH levels actually prevented the cytolethality of combined MMAs(V) and exogenous GSH. These findings indicate that human arsenic metabolite MMAs(V) is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects.
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PMID:Cellular glutathione prevents cytolethality of monomethylarsonic acid. 1499 80

BACKGROUND:: Nipradilol (3,4-dihydro-8-[2-hydroxy-3-isopropyl-amino]propoxy-3-nitroxy-2-H-1-benzopyran), a potent non-selective beta-adrenoceptor antagonist, has been shown to increase NO production. The mechanisms are up-regulation of nitric oxide synthase (NOS) and direct release of NO from nipradilol. The process of direct NO release from nipradilol requires a reductase, such as glutathione S-transferase (GST) in some cells but non-enzymatic NO release was reported in pig coronary arteries. Direct NO release from nipradilol in human coronary arteries has not been examined yet, though this information is of importance. PURPOSE:: To demonstrate direct NO release from nipradilol in human coronary arterial smooth muscle cells (HCASMC) by using a fluorescent NO probe (DAF-2) and an NO-electrode. METHODS AND RESULTS:: HCASMC were loaded with DAF-2 and images of fluorescence (515nm) were obtained under excitation at 488nm through an intensified CCD with an inverted phase-contrast microscope. Concomitantly, NO was measured using an NO-electrode (0.2mm o.d.; 501, Inter Medical Co. Ltd., Nagoya, Japan) after addition of various concentrations of nipradilol (1, 5 or 10microM) with or without ethacrynic acid (GST inhibitor). The cells showed no fluorescence at baseline, but intense fluorescence appeared at 30min after addition of 10microM nipradilol. The intensities of fluorescence at 30min in the control, nipradilol and nipradilol with ethacrynic acid groups were 98 +/- 6, 163 +/- 10 and 128 +/- 6% of the baseline level, respectively. Ethacrynic acid itself did not affect the fluorescence. Continuous measurements of NO by the electrode showed the NO generation peaked at about 30min, remained at the same level till about 45min and then gradually declined. Nipradilol did not produce NO at all in the absence of cells. The dose-dependency study of NO release from nipradilol showed 45 +/- 12, 72 +/- 24 and 157 +/- 23nM, respectively, at 1, 5 and 10microM nipradilol. All experiments were performed under conditions where endogenous formation of NO was inhibited by an NOS inhibitor (10(-4)M N(G)-monomethyl-l-arginine (l-NMMA)). CONCLUSION:: Nipradilol can release NO in the presence of human coronary arterial smooth muscle cells and the denitration reaction catalyzed by a reductase such as glutathione S-transferase contributes substantially to NO release from nipradilol.
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PMID:Direct nitric oxide release from nipradilol in human coronary arterial smooth muscle cells observed with fluorescent NO probe and NO-electrode. 1536 17

Ethacrynic acid (EA), an alpha,beta-unsaturated carbonyl compound, is a glutathione S-transferase P1-1 (GSTP1-1) inhibitor. Twenty-one novel EA derivatives have been synthesized. The effects of these compounds on GSTP1-1 activity and on the proliferation of human leukemia HL-60 cells have been determined. Compounds with a halogen substitution at the 3'-position of the aromatic ring have greater inhibitory effects on GSTP1-1 activity than those of compounds with a methyl substitution there. Compounds with substitutions at both the 2'- and 3'-positions of the aromatic ring have more antiproliferative ability than those with one substitution at 3'-position. Esterification of the carboxyl group appears to increase the antiproliferative ability.
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PMID:The synthesis of alpha,beta-unsaturated carbonyl derivatives with the ability to inhibit both glutathione S-transferase P1-1 activity and the proliferation of leukemia cells. 1728 20


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