Inhibition Of Glutathione S-transferase Research Articles

Inhibition of Glutathione S-Transferase by Ethacrynic Acid Augments Ischemia-Reperfusion Damage and Apoptosis and Attenuates the Positive Effect of Ischemic Postconditioning in a Bilateral Acute Hindlimb Ischemia Rat Model

Aims: We studied the effects of the inhibition of the endogene antioxidant glutathione-S-transferase (GST) by ethacrynic acid (EA) on ischemia-reperfusion (IR) injury and postconditioning (PC) in the lower extremities. We aimed to examine the oxidative stress parameters (OSP), inflammatory response and activation of proapoptotic signaling proteins (PSP) after revascularization surgery. Methods: Sixty Wistar rats were divided into 6 groups: control, IR, PC, EA-control, IR and administration of EA (IR/EA) and PC and administration of EA (PC/EA). The IR, PC, IR/EA and PC/EA groups underwent 60 min of infrarenal aortic cross-clamping. After that, PC was performed in the PC and PC/EA groups. In 3 of the groups, the animals were treated with EA (EA-control, IR/EA and PC/EA groups) as well. The ischemia was followed by 120 min of reperfusion. Blood samples and biopsy specimens were collected from the quadriceps muscle. Plasma malondialdehyde, reduced glutathione, thiol/sulfhydryl group levels, TNF-α and IL-6 concentrations and superoxide-dismutase enzyme activity were measured. Results: The levels of the OSP and the inflammatory proteins were higher in the EA-administered groups. The ratio of phosphorylated PSP was higher in the EA-administered groups and the protective effect of PC did not develop. Conclusions: Inhibition of GST by EA augmented the IR damage. GST inhibition was associated with a different activation of the mitogen-activated protein kinases and the PSP, regulating these pathways in the process of apoptosis and PC.

A practical fluorogenic substrate for high-throughput screening of glutathione S-transferase inhibitors.

We report a new fluorogenic substrate for glutathione S-transferase (GST), 3,4-DNADCF, enabling the assay with a low level of nonenzymatic background reaction. Inhibitors against Noppera-bo/GSTe14 from Drosophila melanogaster were identified by high throughput screening using 3,4-DNADCF, demonstrating the utility of this substrate.

15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways

Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic “inactivation” of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L.laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L.laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1.

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

Novel enzyme inhibiting natural products from medicinally important plants

Natural products derived from plants, marine organisms and microorganisms exhibit interesting anti-microbial, anti-viral, and anti-inflammatory activities. These bioactivities make natural products an important source for the discovery of new pharmaceutical agents, and more than 60% of the drugs available on the market are of natural product origin. One of the aspects of drug discovery process is the identification of small molecules with enzyme-inhibiting activities. Enzymes are essential to human life, mediating biochemical processes including metabolism, cellular signal transduction, cell cycling, and development. Malfunction in these biochemical systems often leads to disease that can be caused either by the dysfunction, overexpression, or hyper-activation of the enzymes involved. An understanding of diseases at the molecular level has provided several enzyme inhibitors in clinics. For instance, galanthamine, a potent acetylcholinesterase (AChE) inhibitor, is used to treat Alzheimer's disease. In-vitro enzyme inhibition assays can easily be performed in any natural product chemistry lab in order to discover lead compounds against various biological targets. For instance, glutathione S-transferase (GST) inhibitors have applications in overcoming the drug resistance problems in cancer and parasite chemotherapy. Fatty acid synthase inhibitors are used to discover anti-malarial, anti-parasitic, anti-TB and anti-fungal compounds. We are involved in discovering new enzyme inhibitors from medicinally important plants. These efforts have resulted in the identification of a few novel AChE, GST and α-glucosidase inhibitors. During this presentation, structure elucidation of these new enzyme inhibitors and their bioactivity data will be discussed. Additionally, results obtained from structure-activity relationship studies will also be presented.

Effects of short-term exposure to fluoxetine and carbamazepine to the collembolan Folsomia candida

Pharmaceuticals, emerging environmental contaminants, have their ecotoxicological effects to non-target organisms in soil largely unknown. This study assessed short-term effects of two human pharmaceuticals, carbamazepine and fluoxetine, to Folsomia candida. Avoidance to spiked soils was assessed after 48 and 96h exposure and biochemical changes (acetylcholinesterase and glutathione S-transferase activities, and lipid peroxidation levels) after 96h. F. candida avoided soils spiked with 0.04, 0.4 and 4mg carbamazepine kg−1 after 48h. However, higher number of organisms were found in soils with 40mg carbamazepine kg−1, a behavior also displayed for 40mg fluoxetine kg−1 spiked soils. After 96h, F. candida showed avoidance behavior to soils with 4 and 40mg carbamazepine kg−1. Acetylcholinesterase activity decreased in 0.4mg fluoxetine kg−1 exposed organisms. Peroxidative damages were detected in organisms exposed to 4 and 40mgkg−1 carbamazepine and glutathione S-transferase inhibition was observed at 40mgkg−1. Data suggests that carbamazepine and fluoxetine may pose risk to soil collembolan.

In vitro inhibition of glutathione S-transferases by several insecticides and allelochemicals in two moth species

Glutathione S-transferases (GSTs) are important detoxification enzymes in insects. This study examined the inhibitory effects of five insecticides (beta-cypermethrin, fenpropathrin, phoxim, abamectin, acetamiprid) and two plant-derived allelochemicals (quercetin, tannic acid) on GST activity in Micromelalopha troglodyta (Graeser) and Clostera anachoreta (Fabricius). GST activities were suppressed by all the insecticides and allelochemicals tested; the two allelochemicals were the most potent inhibitors, reducing GST activity in vitro in a dosage-dependent manner. Kinetic analyses of GST inhibition by quercetin and tannic acid in M. troglodyta and C. anachoreta suggested biochemical differences in their GSTs.

Apoptosis and Differentiation of K562 Cells by Targeting GST-O1 to Inhibit 4-HNE Metabolism

Bcr-Abl kinase inhibitors are very effective drugs for treatment of chronic myelogenous leukemia (CML), but treatment options are limited and relatively ineffective for patients with de-novo or acquired resistance. The K562 human erythroleukemia cell line, derived from a pleural effusion during blast crisis of a CML patient, is very useful for studying hematopoietic differentiation because it undergoes differentiation and apoptosis in response to chemicals that propagate lipid-peroxidation. 4-hydroxynonenal (4-HNE), a reactive aldehyde produced from peroxidation of polyunsaturated fatty acids, is metabolized primarily by glutathione S-transferases (GSTs). 4-HNE causes differentiation, apoptosis and necrosis in K562 cells, but cannot be used as a drug for resistant CML because of its highly toxic nature. Present studies addressed the possibility of developing an alternative targeted treatment aimed at increasing intracellular 4-HNE through inhibition of GST. Because the major GST-isoenzymes in leukemia cells are also present in normal tissues, we explored the possibility of modulating cellular 4-HNE levels by inhibiting GST isozymes with high activity towards 4-HNE. Our studies identified the presence of the GSTO1 isoenzyme in K562 cells, demonstrated its activity towards 4-HNE, and showed that its depletion causes apoptosis, necrosis and differentiation of these cells. These effects of GSTO1 depletion appear to involve RUNX1 mediated transcriptional regulation of GM-CSF. These findings offer a new target for treatment of resistant CML.

Inhibitory effects of purpurogallin on the endothelial protein C receptor shedding in vitro and in vivo

Endothelial cell protein C receptor (EPCR) plays important roles in the regulation of blood coagulation and inflammation. Activity of EPCR is markedly changed by ectodomain cleavage and released as soluble protein (sEPCR). EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). Purpurogallin (PPG) plays an important role in inhibiting glutathione S-transferase and xanthine oxidase as well as effective in the cell protection of several cell types. Here, we investigated the effects of PPG on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Human umbilical vein endothelial cells pretreated with PPG (0, 5, 10, 20 or 50 μg/mL) for 6 h and exposed to PMA (1 μM) for 1 h, and CLP-operated mice were administrated with PPG. Data showed that treatment with PPG resulted in potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. In addition, PPG reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential for use of PPG as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

Ethacrynic Acid Oxadiazole Analogs Induce Apoptosis in Malignant Hematologic Cells through Downregulation of Mcl-1 and c-FLIP, Which Was Attenuated by GSTP1-1

Ethacrynic acid, a diuretic, inhibits glutathione S-transferase P1-1 (GSTP1-1) activity and induces cell death in malignant cells at high concentrations. To improve ethacrynic acid activity, ethacrynic acid oxadiazole analogs 6s and 6u were synthesized. Although both compounds have greater antiproliferative effects than ethacrynic acid in human HL-60 cells, 6u has a reduced ability to inhibit GSTP1-1 activity. The mechanisms of both 6s- and 6u-induced cell death as well as the role of GSTP1-1 in their actions were studied. Both 6s and 6u equally induced apoptosis in HL-60 cells due to the activation of caspase-3, -9, and -8, which was correlated with the downregulation of antiapoptotic proteins c-FLIP, Mcl-1, and XIAP. The caspase inhibitor Z-VAD-FMK blocked the reduction of XIAP, but not of c-FLIP and Mcl-1, in 6s-treated cells. The reduction of c-FLIP and Mcl-1 by 6s was not blocked by the proteasomal inhibitor MG132, but was correlated with inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK) and eIF4E. Both 6s and 6u decreased the intracellular glutathione (GSH) levels. N-acetylcysteine blocked reduction in the levels of Mcl-1, c-FLIP, and intracellular GSH as well as apoptosis in HL-60 cells treated by either compound. Silencing of GSTP1-1 in K562 cells sensitized, but overexpression of GSTP1-1 in Raji cells blocked, apoptosis induction by either compound. GSH conjugation at the methylene group abrogated the ability of inducing apoptosis. These data suggest that the methylene group plays an important role in the downregulation of c-FLIP and Mcl-1 proteins and apoptosis induction, which is inactivated by GSTP1-1 by forming GSH conjugates.

English

Essential oil extracted from plants has been widely investigated for pest control properties, with some proving to be toxic in insect pests. In this study, effect of the synergist diethyl maleate (DEM) on toxicity of one monoterpenoids, linalool was studied against two most common stored-product insects such as Callosobruchus maculatus and Rhyzopertha dominica . Diethyl maleate was combined in mass ratios (1:8 and 1:4) with acetone used and applied on C. maculatus and R. dominica adult. Five concentrations of linalool were tested with four replications at 24 and 48 h with 30 adult insect in each replication. After 24 h of exposure, the LC 50 values were estimated to be 23.61 and 31.01 μl/l air, and after 48 h, they were 15.07 and 21.84 for each insect, respectively. A combination of inalool with the synergist after 24 h of exposure, the LC 50 values was estimated to be 11.93 and 13.07 μl/l air and after 48 h, they were 7.38 and 7.93, respectively for each insect. The synergist is able to block the specific system of enzymes involved in selection of tolerance in susceptible generations. Diethyl maleate is an inhibitor of glutathione S-transferase (GST) activity. These results show that diethyl maleate decrease doses of linalool. Key words : Diethyl maleate, linalool, LC50, synergism, Rhyzopertha dominica, Callosobruchus maculatus.

Diphenyl diselenide prevents hepatic alterations induced by paraquat in rats

This study aimed to investigate the beneficial effect of diphenyl diselenide (PhSe)2 on paraquat (PQ) induced alterations in rats liver. Adult male Wistar rats received (PhSe)2 at 10mgkg−1, by oral administration (p.o.), during five consecutive days. Twenty-four hours after the last (PhSe)2 dose, rats received PQ at 15mgkg−1, in a single intraperitoneally injection (i.p.). Seventy-two hours after PQ exposure, animals were sacrificed by decapitation for blood and liver samples obtainment. Histological alterations induced by PQ exposure, such as inflammatory cells infiltration and edema, were prevented by (PhSe)2 administration. Moreover, (PhSe)2 prevented hepatic lipid peroxidation (LPO) induced by PQ and was effective in reducing the myeloperoxidase (MPO) activity in liver, which was enhanced by PQ exposure. (PhSe)2 also was effective in protecting against the reduction in ascorbic acid and non-protein thiols (NPSH) levels induced by PQ. The inhibition of glutathione S-transferase (GST) activity, in rats exposed to PQ, was normalized by (PhSe)2 pre-treatment, whereas the inhibition of catalase (CAT) activity was not prevented by (PhSe)2. The serum alkaline phosphatase (ALP) inhibition, induced by PQ administration, was also prevented by (PhSe)2 pre-treatment. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not modified by PQ and/or (PhSe)2 administration. Therefore, (PhSe)2 pre-treatment was effective in protecting against the hepatic alterations induced by PQ in rats. This protective effect can involve the antioxidant and anti-inflammatory properties of (PhSe)2.

The role of the inhibition of glutathione-S-transferase in the protective mechanisms of ischemic postconditioning

The antioxidant glutathione-S-transferase (GST) is a crucial determinant of the development of ischaemic-reperfusion (I/R) injury, and plays a pivotal role in the regulation of the mitogen activated protein kinase (MAPK) pathways involved in stress response and apoptosis. The aim of this study was to investigate whether inhibition of GST can abolish the benefit of ischaemic postconditioning (IPoC). A neonatal rat cardiomyocyte cell culture was prepared and divided into 6 groups: (I) control group without treatment; (II) cells exposed to simulated I/R; (III) simulated I/R (sI/R) with IPoC; (IV) ethacrynic acid (EA) alone; (V) sI/R with EA; and (VI) sI/R and IPoC together with EA. Viability of the cells was measured by MTT assay, the quantity of apoptotic cells was assessed by flow cytometry following annexin V-FITC - propidium-iodide double staining. The activation of JNK, p38, ERK/p42-p44 MAPKs, and GSK-3β protein kinase was determined by flow-cytometric assay. GST inhibition markedly increased the apoptosis and decreased the cell viability despite IPoC. The protective effect of IPoC was lost in GST-inhibited groups for all MAPKs and GSK-3β. GST activity is required for the survival of cultured cardiomyocytes under stress conditions. GST inhibition was associated with differential activation of MAP and the protein kinases regulating these pathways in the process of ischaemic postconditioning.

Cantharidin Impedes Activity of Glutathione S-Transferase in the Midgut of Helicoverpa armigera Hübner

Previous investigations have implicated glutathione S-transferases (GSTs) as one of the major reasons for insecticide resistance. Therefore, effectiveness of new candidate compounds depends on their ability to inhibit GSTs to prevent metabolic detoxification by insects. Cantharidin, a terpenoid compound of insect origin, has been developed as a bio-pesticide in China, and proves highly toxic to a wide range of insects, especially lepidopteran. In the present study, we test cantharidin as a model compound for its toxicity, effects on the mRNA transcription of a model Helicoverpa armigera glutathione S-transferase gene (HaGST) and also for its putative inhibitory effect on the catalytic activity of GSTs, both in vivo and in vitro in Helicoverpa armigera, employing molecular and biochemical methods. Bioassay results showed that cantharidin was highly toxic to H. armigera. Real-time qPCR showed down-regulation of the HaGST at the mRNA transcript ranging from 2.5 to 12.5 folds while biochemical assays showed in vivo inhibition of GSTs in midgut and in vitro inhibition of rHaGST. Binding of cantharidin to HaGST was rationalized by homology and molecular docking simulations using a model GST (1PN9) as a template structure. Molecular docking simulations also confirmed accurate docking of the cantharidin molecule to the active site of HaGST impeding its catalytic activity.

Cefadroxil potency as cancer co-therapy candidate by glutathione s-transferase mechanism

Background: Glutathione S-transferases (GSTs) have an important role in the detoxification of electrophiles, such as some anticancer drugs. Compounds with phenolic and/or α,b-unsaturated carbonyl group have been known as GSTs inhibitor in vitro. Cefadroxil in vitro decreased GST-Pi activity but not GSTs in rat kidney cytosol. GST inhibitor in a specific organ and of a specific class is needed for safety in cancer chemotherapy. The study aims to observe the effect of cefadroxil on GSTs in vivo in rat kidney cytosol and then compare it to those seen for liver, lung, and spleen in vivo.

Synthesis and Biological Study of Novel Indole-3-Imine-2-on Derivatives as Src Kinase and Glutathione S-Transferase Inhibitors

Synthesis and Biological Study of Novel Indole-3-Imine-2-on Derivatives as Src Kinase and Glutathione S-Transferase Inhibitors

Coniferyl Ferulate, a Strong Inhibitor of Glutathione S-Transferase Isolated from RadixAngelicae sinensis, Reverses Multidrug Resistance and Downregulates P-Glycoprotein

Glutathione S-transferase (GST) is the key enzyme in multidrug resistance (MDR) of tumour. Inhibition of the expression or activity of GST has emerged as a promising therapeutic strategy for the reversal of MDR. Coniferyl ferulate (CF), isolated from the root of Angelica sinensis (Oliv.) Diels (Radix Angelicae sinensis, RAS), showed strong inhibition of human placental GST. Its 50% inhibition concentration (IC50) was 0.3 μM, which was greater than a known GSTP1-1 inhibitor, ethacrynic acid (EA), using the established high-throughput screening model. Kinetic analysis and computational docking were used to examine the mechanism of GST inhibition by CF. Computational docking found that CF could be fully docked into the gorge of GSTP1-1. The further exploration of the mechanisms showed that CF was a reversible noncompetitive inhibitor with respect to GSH and CDNB, and it has much less cytotoxicity. Apoptosis and the expression of P-gp mRNA were evaluated in the MDR positive B-MD-C1 (ADR+/+) cell line to investigate the MDR reversal effect of CF. Moreover, CF showed strong apoptogenic activity and could markedly decrease the overexpressed P-gp. The results demonstrated that CF could inhibit GST activity in a concentration-dependent manner and showed a potential MDR reversal effect for antitumour adjuvant therapy.

Calixarene Phosphonous Acids: Synthesis and Biological Activity

The cone shaped сalix[4]arene hydroxyphosponous, ketophosphonous, methylenebisphosphonous, and hydroxymethylenebisphosphonous acids are specific inhibitors of glutathione S-transferase, ATPases of smooth muscle cells, fibrin polymerization processes

Glutathione S‐transferase P1‐1 as a target for mesothelioma treatment

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione-S-transferase, which catalyzes the conjugation between glutathione and platinum(II)-containing drugs. Therefore, we evaluated the effect of the strong glutathione S-transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO-211H, MPP89, MM-B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis. NBDHEX also caused severe oxidative stress in the MPP89 cells and, to a lesser extent, in the MMB1 cells, while it did not cause a significant redox imbalance in the other cell lines. The efficacy of the drug was found to be comparable or even higher than that of cisplatin. Moreover, it showed synergistic or additive effects when used in combination with cisplatin. In conclusion, NBDHEX was effective on mesothelioma cell lines, with IC(50) values in the low micromolar range (IC(50) between 1 and 4 μM). These findings indicate that NBDHEX, alone or in combination with cisplatin, is a promising new strategy for treating this rare and aggressive malignancy.