Gamma-glutamyl Transpeptidase Inhibitor Research Articles

Antihelminthic effect of thymoquinone against biliary amphistome, Gigantocotyleexplanatum

Recent research on the emergence of parasitic resistance to commonly prescribed anthelmintics has sparked a greater interest in finding novel therapeutic molecules, including those derived from plants. The use of medicinal plants and their derivatives has been viewed as an alternative source of anti-parasitic compounds and as being safe in comparison to synthetic medications due to the absence of adverse effects, ease of accessibility, and little to no expense.Consequently, in the current study, thymoquinone (TQ), an active component of Nigella sativa (Black cumin), has been tested to see their effect on the activity of some important parameters of Gigantocotyle explanatum worms, including Gamma-glutamyl Transpeptidase (GGT), glutathione (GSH), Glutathione-S-Transferase (GST), Superoxide dismutase (NO). Additionally, various other survival indicators are also used, such as assays for motility, tegument damage, and DNA fragmentation.G. explanatum adult flukes were in vitro treated to thymoquinone at various concentrations for 3 h at 37 °C. Even though all of the worms were still alive after 3 h of exposure, there was a substantial (p < 0.05) reduction in worm motility at a concentration of 90 M. There were pronounced tegumental disturbances, a loss of surface annulations, and erosion in the papillae posterior region and around the acetabulum. A significant (p < 0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed. A significant inhibition of Gamma-glutamyl Transpeptidase (GGT) in thymoquinone treated worms was also evident. Thymoquinone and GGT also displayed a high interaction during in silico molecular docking, suggesting that this combination may be more effective at inhibiting the antioxidant enzymes of G. explanatum. The present findings suggest that thymoquinone would reduce the worm capacity for detoxification, while GGT inhibition would have a major impact on their ability to transport amino acids across the tegument. Thymoquinone thus seemed to be a promising anthelmintic compound for future investigations.

Design and evaluation of novel analogs of 2-amino-4-boronobutanoic acid (ABBA) as inhibitors of human gamma-glutamyl transpeptidase

Inhibitors of gamma-glutamyl transpeptidase (GGT1, aka gamma-glutamyl transferase) are needed for the treatment of cancer, cardiovascular illness and other diseases. Compounds that inhibit GGT1 have been evaluated in the clinic, but no inhibitor has successfully demonstrated specific and systemic GGT1 inhibition. All have severe side effects. L-2-amino-4‑boronobutanoic acid (l-ABBA), a glutamate analog, is the most potent GGT1 inhibitor in vitro. In this study, we have solved the crystal structure of human GGT1 (hGGT1) with ABBA bound in the active site. The structure was interrogated to identify interactions between the enzyme and the inhibitor. Based on these data, a series of novel ABBA analogs were designed and synthesized. Their inhibitory activity against the hydrolysis and transpeptidation activities of hGGT1 were determined. The lead compounds were crystalized with hGGT1 and the structures solved. The kinetic data and structures of the complexes provide new insights into the critical role of protein structure dynamics in developing compounds for inhibition of hGGT1.

Amlodipine alleviates cisplatin-induced nephrotoxicity in rats through gamma-glutamyl transpeptidase (GGT) enzyme inhibition, associated with regulation of Nrf2/HO-1, MAPK/NF-κB, and Bax/Bcl-2 signaling

BackgroundThe therapeutic utility of the effective chemotherapeutic agent cisplatin is hampered by its nephrotoxic effect. We aimed from the current study to examine the possible protective effects of amlodipine through gamma-glutamyl transpeptidase (GGT) enzyme inhibition against cisplatin nephrotoxicity. MethodsAmlodipine (5 mg/kg, po) was administered to rats for 14 successive days. On the 10th day, nephrotoxicity was induced by a single dose of cisplatin (6.5 mg/kg, ip). On the last day, blood samples were collected for estimation of kidney function, while kidney samples were used for determination of GGT activity, oxidative stress, inflammatory, and apoptotic markers, along with histopathological evaluation. ResultsAmlodipine alleviated renal injury that was manifested by significantly diminished serum creatinine and blood urea nitrogen levels, compared to cisplatin group. Amlodipine inhibited GGT enzyme, which participates in the metabolism of extracellular glutathione (GSH) and platinum-GSH-conjugates to a reactive toxic thiol. Besides, amlodipine diminished mRNA expression of NADPH oxidase in the kidney, while enhanced the anti-oxidant defense by activating Nrf2/HO-1 signaling. Additionally, it showed marked anti-inflammatory response by reducing expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB), with subsequent down-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1). Moreover, amlodipine reduced Bax/Bcl-2 ratio and elevated hepatocyte growth factor (HGF), thus favoring renal cell survival. ConclusionsEffective GGT inhibition by amlodipine associated with enhancement of anti-oxidant defense and suppression of inflammatory signaling and apoptosis support our suggestion that amlodipine could replace toxic GGT inhibitors in protection against cisplatin nephrotoxicity.

Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase.

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.

Therapeutic Effect of GGsTop, Selective Gamma-glutamyl Transpeptidase Inhibitor, on a Mouse Model of 5-Fluorouracil-induced Oral Mucositis

Oral mucositis (OM) induced by cancer chemotherapy has a high incidence and serious symptoms, which often force chemotherapy to be stopped. GGsTop is a newly-discovered gamma-glutamyl transpeptidase (GGT) inhibitor. Previous research suggested that inhibition of GGT suppressed reactive oxygen species and induced the production of collagen and elastin. We hypothesized that GGsTop could safely treat OM. A mouse model of OM was treated with GGsTop and ulcer area, weight, and white blood cell count were determined. The treatment effect was also evaluated by hematoxylin-eosin and collagen staining. The therapeutic effect of GGsTop was better than that of an existing drug and may be safely used in combination with chemotherapy. Furthermore, GGsTop promoted collagen production in oral mucosa. GGsTop treated OM quickly and safely. GGsTop is highly valuable for use as a treatment for OM.

"Phylogenetic and evolutionary analysis of functional divergence among Gamma glutamyl transpeptidase (GGT) subfamilies".

Backgroundγ-glutamyltranspeptidase (GGT) is a bi-substrate enzyme conserved in all three domains of life. It catalyzes the cleavage and transfer of γ-glutamyl moiety of glutathione to either water (hydrolysis) or substrates like peptides (transpeptidation). GGTs exhibit great variability in their enzyme kinetics although the mechanism of catalysis is conserved. Recently, GGT has been shown to be a virulence factor in microbes like Helicobacter pylori and Bacillus anthracis. In mammalian cells also, GGT inhibition prior to chemotherapy has been shown to sensitize tumors to the therapy. Therefore, lately both bacterial and eukaryotic GGTs have emerged as potential drug targets, but the efforts directed towards finding suitable inhibitors have not yielded any significant results yet. We propose that delineating the residues responsible for the functional diversity associated with these proteins could help in design of species/clade specific inhibitors.ResultsIn the present study, we have carried out phylogenetic analysis on a set of 47 GGT-like proteins to address the functional diversity. These proteins segregate into various subfamilies, forming separate clades on the tree. Sequence conservation and motif prediction studies show that even though most of the highly conserved residues have been characterized biochemically in previous studies, a significant number of novel putative sites and motifs are discovered that vary in a clade specific manner. Many of the putative sites predicted during the functional divergence type I and type II analysis, lie close to the known catalytic residues and line the walls of the substrate binding cavity, reinforcing their role in modulating the substrate specificity, catalytic rates and stability of this protein.ConclusionThe study offers interesting insights into the evolution of GGT-like proteins in pathogenic vs. non-pathogenic bacteria, archaea and eukaryotes. Our analysis delineates residues that are highly specific to each GGT subfamily. We propose that these sites not only explain the differences in stability and catalytic variability of various GGTs but can also aid in design of specific inhibitors against particular GGTs. Thus, apart from the commonly used in-silico inhibitor screening approaches, evolutionary analysis identifying the functional divergence hotspots in GGT proteins could augment the structure based drug design approaches.ReviewersThis article was reviewed by Andrei Osterman, Christine Orengo, and Srikrishna Subramanian. For complete reports, see the Reviewers’ reports sectionElectronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0080-7) contains supplementary material, which is available to authorized users.

Abstract 4546: Depleting cysteine: A novel approach to tumor therapy

Abstract A cure remains elusive for many types of tumors that initially respond to chemotherapy but recur with drug-resistant disease. Cysteine, (or its oxidized form cystine) is an essential amino acid for tumors. Enhanced uptake of cysteine and/or cystine is required for tumors to maintain their intracellular glutathione (GSH) levels and redox balance. In addition, some stem cells within epithelial tumors express CD44v8-10, which stabilizes the cystine transporter, increasing cystine uptake into the cell and enhancing resistance to pro-oxidant therapy. Depleting cysteine during treatment has a profound effect on sensitizing the tumor to therapy. We have a novel strategy that reduces the cysteine (and cystine) concentration in serum and extracellular fluid. The strategy exploits the finding that a severe cysteine deficiency develops in mice lacking the enzyme gamma-glutamyl transpeptidase (GGT). This enzyme reclaims the cysteine from GSH as the blood is filtered through the kidney. Inhibiting GGT activity results in excretion of GSH in the urine and a precipitous drop in the concentration of cysteine and cystine in serum. The inhibitors of GGT that have been evaluated in the clinic are glutamate analogs and are too toxic for clinical use. We have discovered and are developing a new class of GGT inhibitors that are not glutamate analogs. These inhibitors are orders of magnitude less toxic than the glutamate analogs. We have determined the crystal structure of human GGT. In the current study we have co-crystallized human GGT with a series of inhibitors. We report the identification of residues within the active site that are critical for inhibitor binding. We have identified changes in the conformation of the active site upon substrate binding. We have also delineated movement within the active site during substrate cleavage. Site-directed mutagenesis and functional assays have confirmed the role of the key residues identified in the structural studies. These data provide new insights for continued development of a potent, specific, non-toxic inhibitor of GGT that can be used clinically in cancer therapy. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Anthony W. G. Burgett, Nancy Wakeham, Simon S. Terzyan. Depleting cysteine: A novel approach to tumor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2015-4546

Leukotriene C4 induces bronchoconstriction and airway vascular hyperpermeability via the cysteinyl leukotriene receptor 2 in S-hexyl glutathione-treated guinea pigs

Cysteinyl leukotrienes act through G-protein-coupled receptors termed cysteinyl leukotriene 1 (CysLT1) and cysteinyl leukotriene 2 (CysLT2) receptors. However, little is known about the pathophysiological role of CysLT2 receptors in asthma. To elucidate the possible involvement of CysLT2 receptors in bronchoconstriction and airway vascular hyperpermeability, we have established a novel guinea pig model of asthma. In vitro study confirmed that CHO-K1 cells, expressing guinea pig CysLT2 and CysLT1 receptors are selectively stimulated by LTC4 and LTD4, respectively. However, when LTC4 was intravenously injected to guinea pigs, the resulting bronchoconstriction was fully abrogated by montelukast, a CysLT1 receptor antagonist, indicating rapid metabolism of LTC4 to LTD4 in the lung. We found that treatment with S-hexyl glutathione (S-hexyl GSH), an inhibitor of gamma-glutamyl transpeptidase, significantly increased LTC4 content and LTC4/(LTD4 plus LTE4) ratio in the lung. Under these circumstances, LTC4-induced bronchoconstriction became resistant to montelukast, but sensitive to Compound A, a CysLT2 receptor antagonist, depending on the dose of S-hexyl GSH. Combination with montelukast and Compound A completely abrogated this spasmogenic response. Additionally, we confirmed that LTC4 elicits airway vascular hyperpermeability via CysLT2 receptors in the presence of high dose of S-hexyl GSH as evidenced by complete inhibition of LTC4-induced hyperpermeability by Compound A, but not montelukast. These results suggest that CysLT2 receptors mediate bronchoconstriction and airway vascular hyperpermeability in guinea pigs and that the animal model used in this study may be useful to elucidate the functional role of CysLT2 receptors in various diseases, including asthma.

Abstract 683: Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase

Abstract We have reported that 73% of ovarian tumors express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). Clinical studies have shown a poorer prognosis for ovarian tumors that are GGT-positive compared to GGT-negative tumors. GGT-positive tumors are able to use the glutathione in the interstitial fluid as an additional source of cysteine, the limiting component of intracellular glutathione synthesis in tumors. The concentration of cysteine/cystine in standard tissue culture media is more than 3 times the concentration in interstitial fluid. In this study we show that in studies of cultured cells, the high concentration of cysteine in tissue culture media obscured the role of GGT expression in drug resistance. However, culturing human ovarian tumor cells in tissue culture media containing physiological concentrations of cystine and glutathione demonstrated that the supply of cysteine/cystine regulates intracellular glutathione concentrations. In these studies, inhibition of GGT blocked the use of extracellular glutathione as a source of cysteine and sensitized the tumors to cisplatin, carboplatin and melphalan. Inhibiting GGT in patients would also inhibit GGT in the kidney resulting in the excretion of glutathione in the urine and depletion of cysteine from serum and interstitial fluid. As a result, inhibition of GGT in patients would sensitize both GGT-positive and GGT-negative tumors to pro-oxidant chemotherapy. Clinical application of this therapeutic strategy is limited by the fact that most inhibitors of GGT are glutamate analogs and too toxic for use in humans. We discovered a non-glutamate analog that is an uncompetitive inhibitor of GGT, and are developing more potent, non-toxic GGT inhibitors. We recently published the crystal structure of human GGT. We have now also solved the structure of the substrate-bound enzyme, the form of the enzyme to which our uncompetitive inhibitors bind. Using structure-based drug design and our physiologically relevant tissue culture system we are designing new GGT inhibitors and evaluating them for their ability to sensitize tumors to chemotherapy regimens used in the clinic. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Nancy Wakeham, Stephanie Wickham, Simon S. Terzyan. Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2014-683

PP39 Relative versus absolute measures of the neighbourhood food environment and diet in the ORiEL Study: a geographically weighted regression approach

The pathophysiology of hepatic veno-occlusive disease is poorly understood. These studies were undertaken to determine the initial cellular target and the role of glutathione detoxification of dacarbazine, a toxin implicated in hepatic veno-occlusive disease. Sinusoidal endothelial cells (SECs) and hepatocytes were isolated and plated in culture dishes. Dacarbazine (5-(3,3-dimethyl-triazeno) imidazole-4-carboxamide), 3 and 6 mM, was toxic to SECs but not to hepatocytes. Onset of toxicity occurred between 11 and 12 hr as determined by serial MTT assays and ethidium homodimer dye exclusion. Glutathione detoxification of dacarbazine in SECs was suggested by: (1) depletion of glutathione before onset of toxicity; (2) exacerbation of toxicity by buthionine sulfoximine (BSO) depletion of glutathione; and (3) protection by exogenous glutathione. Protection by exogenous glutathione may be by uptake of intact tripeptide rather than by extracellular hydrolysis: neither acivicin (inhibitor of gamma-glutamyltranspeptidase) nor BSO (inhibitor of gamma-glutamylcysteine synthetase) blocked the protective effect, and glutathione disulfide did not protect. The relative resistance to dacarbazine toxicity seen in hepatocytes is not due to more efficient GSH detoxification, because toxicity was not unmasked in hepatocytes cultures in medium lacking sulfur amino acid precursors of GSH. In conclusion, glutathione status may play an important role in the susceptibility to toxicity. Furthermore, the findings suggest that the SEC is the initial in vivo target of dacarbazine due to a relatively higher level of metabolic activation that more readily overcomes the available detoxification.

Helicobacter pylori–Induced Loss of Survivin and Gastric Cell Viability Is Attributable to Secreted Bacterial Gamma-Glutamyl Transpeptidase Activity

Helicobacter pylori is the etiologic agent of a series of gastric pathologies that may culminate in the development of gastric adenocarcinoma. An initial step in this process is the loss of glandular structures in the gastric mucosa, presumably as the consequence of increased apoptosis and reduced cellular regeneration, which may be attributed to the combination of several bacterial and host factors and to an unfavorable proinflammatory environment. In a previous study, we showed that survivin, a member of the inhibitor of apoptosis protein family, is expressed in the normal human gastric mucosa and that its levels decrease in the mucosa of infected patients and in gastric cells exposed in culture to the bacteria, coincident with increased cell death in the latter case. We investigated the bacterial factors responsible for loss of survivin in gastric cells exposed to H. pylori. The results of this study indicated that the loss of survivin due to H. pylori infection involves proteasome-mediated degradation of the protein. Studies with isogenic mutants deficient in either CagA, VacA, lipopolysaccharide, or gamma-glutamyl transpeptidase (GGT) implicated the latter in H. pylori-induced loss of survivin and cell viability. Moreover, experiments with the GGT inhibitor 6-diazo-5-oxo-l-norleucine and purified recombinant GGT protein indicated that secreted bacterial GGT activity was required and sufficient to induce these effects.

Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells

Ethanol is independently known to cause tissue damage through various mechanisms. This study was designed to evaluate the protective effect of marine microalgae, Navicula incerta protein enzymatic hydrolysates (NEHs) against ethanol-induced hepatotoxicity in HepG2 cells transfected with human CYP2E1. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. When the alcohol-induced cells were treated with NEHs at various concentrations, there was a dose-dependent decrease of gamma-glutamyl transpeptidase (GGT) activity in the culture media and loss of cell viability. Among the NEHs constituents the hydrolysates which were obtained by papain (P-NEH), pronase-E (PR-NEH) and α-chymotrypsin (A-NEH) activity attenuated the ethanol cytotoxicity effectively, respectively. The activity appeared to be a GGT inhibitor. Therefore, the cytoprotective effects at alcohol-induced HepG2/CYP2E1 cells could be attributed to the inhibition of GGT activity by NEHs. This study suggests that NEHs have enough potential to be considered as highly active compounds against ethanol toxicity which leads NEHs to be significant nutraceuticals.

Direct Exposure to Ammonia and Hyperammonemia Increase the Extracellular Accumulation and Degradation of Astroglia-Derived Glutathione in the Rat Prefrontal Cortex

We endeavored here to shed light on the supply of glutathione (GSH) precursors from glial cells to neurons and on the interference of ammonia with this process. Administration of ammonium chloride (ammonia) via a microdialysis probe to the rat prefrontal cortex rapidly increased GSH content in the microdialysates. The increase was abrogated by the inhibitor of astrocytic energy metabolism fluoroacetate and the inhibitor of glutathione synthesis buthionine sulfoximine. GSH in the microdialysates was significantly elevated in rats with simple hyperammonemia (HA) or hepatic encephalopathy (HE) (three ip administrations of ammonium acetate or thioacetamide, respectively, at 24-h intervals), only when microdialysis was carried out in the presence of a gamma-glutamyltranspeptidase (gammaGT) inhibitor acivicin. Extracellular GSH increased in cultured rat cortical astrocytes treated with 5mM ammonia for 1 h, but not for 3-72 h, which was the period of increased gammaGT activity. GSH remained increased during the whole 72-h incubation with 5 or 10mM ammonia in C6 glioma cells, where gammaGT activity is intrinsically low and was not increased by ammonia. Collectively, the results suggest that in rats with HA or HE ammonia specifically promote GSH synthesis and export from astrocytes and increase its extracellular degradation, which may improve the availability of precursors for GSH synthesis in neurons and their resistance to ammonia toxicity.

Abstract 760: Sensitizing tumors to chemotherapy by inhibition of gamma-glutamyl transpeptidase

Abstract Tumors that express the cell surface enzyme gamma-glutamyl transpeptidase (GGT) have increased resistance to chemotherapy and radiation. GGT cleaves the gamma-glutamyl bond of extracellular glutathione, initiating the cleavage of glutathione into glutamate, cysteine and glycine. Glutathione can not be taken up intact by tumor cells. Cleavage of extracellular glutathione increases the supply of cysteine to the cell, which is essential to increasing intracellular glutathione levels. Glutathione detoxifies chemotherapy drugs, and elevated levels of intracellular glutathione can block apoptosis. We are developing inhibitors of GGT that can be used to sensitize tumors to therapy. The standard assay for GGT activity uses p-nitroanilide derivatives as substrates, millimolar concentrations of dipeptides as acceptors and is buffered at pH 8.6. Under these assay conditions several groups of investigators have shown that GGT cleaves the gamma-glutamyl bond of the substrate and transfers the gamma-glutamyl group to an acceptor by a Ping-Pong mechanism. However, the standard assay for GGT activity does not measure the reaction catalyzed by GGT in vivo. We have assayed GGT activity under physiologic conditions. We have found that some compounds that inhibit GGT activity in the standard reaction do not inhibit under physiologic conditions. Our data reveal that, at physiologic pH and osmolarity with reduced concentrations of acceptor, the enzyme does cleave the gamma-glutamyl bond by a Ping-Pong mechanism, but hydrolysis of the intermediate of the gamma-glutamyl enzyme intermediate is significant, and competition between water and the peptide acceptor for the intermediate makes it appear Sequential. Several compounds that inhibit GGT in the standard assay actually accelerate activity as the concentration of the acceptor is reduced to physiologic levels. To identify inhibitors that are optimized for clinical use, we have developed an assay that evaluates GGT cleavage of glutathione, the physiologic substrate, at physiologic pH and osmolarity. We are using our previously reported inhibitor OU749 and other lead compounds to further characterize the physiologic GGT reaction and to identify essential substructural components for inhibition of GGT activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 760.

Mechanistic study of BNP7787-mediated cisplatin nephroprotection: modulation of gamma-glutamyl transpeptidase

The mechanisms for cisplatin-induced renal cell injury have been the focus of intense investigation for many years with a view to provide a more effective and convenient form of nephroprotection. BNP7787 (disodium 2,2'-dithio-bis ethane sulfonate; dimesna, Tavocept), is a water-soluble disulfide investigational new drug that is undergoing clinical development for the prevention and mitigation of clinically important chemotherapy-induced toxicities associated with platinum-type chemotherapeutic agents. We hypothesized that part of BNP7787's mechanism of action (MOA) pertaining to the potential prevention of cisplatin-induced nephrotoxicity involves the inhibition of gamma-glutamyl transpeptidase (GGT) activity, mediated by BNP7787-derived mesna-disulfide heteroconjugates that contain a terminal gamma-glutamate moiety [e.g., mesna-glutathione (MSSGlutathione) and mesna-cysteinyl-glutamate (MSSCE)]. Inhibition studies were conducted on human and porcine GGT to determine the effect of mesna-disulfide heteroconjugates on the enzyme's activity in vitro. These studies utilized a fluorimetric assay that monitored the hydrolysis of L-gamma-glutamyl-7-amino-4-trifluoromethylcoumarin (GG-AFC) to AFC. Mesna-disulfide heteroconjugates that contained gamma-glutamyl moieties were potent inhibitors of human and porcine GGT. An in situ-generated mesna-cisplatin conjugate was not a substrate for GGT. The GGT xenobiotic metabolism pathway is postulated to be a major toxification pathway for cisplatin nephrotoxicity, and BNP7787 may play a novel and critical therapeutic role in the modulation of GGT activity. We further postulate that there are two general mechanisms for BNP7787-mediated nephroprotection against cisplatin-induced nephrotoxicity involving this pathway. First, the active BNP7787 pharmacophore, mesna, produces an inactive mesna-cisplatin conjugate that is not a substrate for the GGT toxification pathway (GGT xenobiotic metabolism pathway) and, second, BNP7787-derived mesna-disulfide heteroconjugates may serve as selective, potent inhibitors of GGT, possibly resulting in nephroprotection by a novel means.

A Novel, Species-specific Class of Uncompetitive Inhibitors of γ-Glutamyl Transpeptidase

Expression of gamma-glutamyl transpeptidase (GGT) in tumors contributes to resistance to radiation and chemotherapy. GGT is inhibited by glutamine analogues that compete with the substrate for the gamma-glutamyl binding site. However, the glutamine analogues that have been evaluated in clinical trials are too toxic for use in humans. We have used high throughput screening to evaluate small molecules for their ability to inhibit GGT and have identified a novel class of inhibitors that are not glutamine analogues. These compounds are uncompetitive inhibitors, binding the gamma-glutamyl enzyme complex. OU749, the lead compound, has an intrinsic K(i) of 17.6 microm. It is a competitive inhibitor of the acceptor glycyl-glycine, which indicates that OU749 occupies the acceptor site while binding to the gamma-glutamyl substrate complex. OU749 is more than 150-fold less toxic than the GGT inhibitor acivicin toward dividing cells. Inhibition of GGT by OU749 is species-specific, inhibiting GGT isolated from human kidney with 7-10-fold greater potency than GGT isolated from rat or mouse kidney. OU749 does not inhibit GGT from pig cells. Human GGT expressed in mouse fibroblasts is inhibited by OU749 similarly to GGT from human cells, which indicates that the species specificity is determined by differences in the primary structure of the protein rather than species-specific, post-translational modifications. These studies have identified a novel class of inhibitors of GGT, providing the basis for further development of a new group of therapeutics that inhibit GGT by a mechanism distinct from the toxic glutamine analogues.

Crystal Structure of Acivicin-Inhibited γ-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis

Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) is a general gamma-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used gamma-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k(max) = 0.033 +/- 0.006 s(-1) and K(I) = 19.7 +/- 7.2 microM. Structure determination of acivicin-modified HpGT (1.7 A; R(factor) = 17.9%; R(free) = 20.8%) demonstrates that acivicin is accommodated within the gamma-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

Treatment With Anti-γ-Glutamyl Transpeptidase Antibody Attenuates Osteolysis in Collagen-Induced Arthritis Mice

The effectiveness of a new antibody treatment on arthritis-associated osteolysis was studied by using CIA mice. GGT, a newly identified bone-resorbing factor, was upregulated in arthritic joints. We generated monoclonal antibodies against GGT and injected them into CIA mice. Mice treated with antibodies showed a reduction in osteoclast number and bone erosion. Gamma-glutamyl transpeptidase (GGT) acts as a bone-resorbing factor that stimulates osteoclast formation. GGT expression has been detected in active lymphocytes that accumulate at inflammation sites, such as rheumatoid arthritis (RA). We hypothesize that GGT is an effective target for suppression of arthritis-related osteoclastogenesis and joint destruction. Here, we describe the therapeutic effect of neutralizing antibodies against GGT on joint destruction using a collagen-induced arthritis (CIA) mouse model. GGT expression in the synovium of RA patients and CIA mice was determined by immunohistochemistry and RT-PCR. Monoclonal antibodies were generated against recombinant human GGT (GGT-mAbs) using BALB/c mice. Antibody treatment was performed by intraperitoneal injections of GGT-mAbs into CIA mice. Effects of antibody treatment on arthritis and bone erosion were evaluated by incidence score, arthritis score, and histopathological observations. The role of GGT in osteoclast development was examined by using the established osteoclastogenic culture system. GGT expression was significantly upregulated in inflamed synovium. Immunohistochemistry revealed that GGT was present in lymphocytes, plasma cells, and macrophages, as well as capillaries. Injection of GGT-mAbs significantly decreased the number of osteoclasts and attenuated the severity of joint destruction in CIA mice. In vitro examination showed that GGT enhanced RANKL-dependent osteoclast formation. GGT stimulated the expression of RANKL in osteoblasts and its receptor RANK in osteoclast precursors, respectively. This study indicates that inflamed synovial tissue-derived GGT acts as a risk factor for joint destruction and that the antibody-mediated inhibition of GGT significantly decreases osteoclast number and bone erosion in CIA mice. GGT antagonists might be novel therapeutic agents for attenuating joint destruction in RA patients.

Akt-mediated activation of HIF-1 in pulmonary vascular endothelial cells by S-nitrosoglutathione.

S-nitrosoglutathione (GSNO) stabilizes the alpha-subunit of hypoxia inducible factor-1 (HIF-1) in normoxic cells, but not in the presence of PI3K inhibitors. In this report, the biochemical pathway by which GSNO alters PI3K/Akt activity to modify HIF-1 expression was characterized in Cos cells and primary pulmonary vascular endothelial cells. GSNO increased Akt kinase activity--and downstream HIF-1alpha protein accumulation and DNA-binding activity--in a dose- and time-dependent manner. The PI3K inhibitors, wortmannin and LY294002, blocked these responses. Neither glutathione nor 8-bromo-cyclic GMP mimicked the GSNO-induced increases in Akt kinase activity. GSNO-induced Akt kinase activity and downstream HIF-1alpha stabilization were blocked by acivicin, an inhibitor of gamma-glutamyl transpeptidase (gammaGT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1alpha stabilization. Moreover, the 3'-phosphatase of phosphoinositides, PTEN (phosphatase and tensin homolog deleted on chromosome ten) was S-nitrosylated by GSNO in pulmonary arterial endothelial cells, which was reversed by dithiothreitol and ultraviolet light. Interestingly, the abundance of S-nitrosylated PTEN also correlated inversely with PTEN activity. Taken together, these results suggest that GSNO induction of Akt appears to be mediated by S-nitrosylation chemistry rather than classic NO signaling through guanylate cyclase/cGMP. We speculate that gammaGT-dependent activation of Akt and subsequent activation of HIF-1 in vascular beds may be relevant to the regulation of HIF-1-dependent gene expression in conditions associated with oxyhemoglobin deoxygenation, as opposed to profoundly low Po(2), in the pulmonary vasculature.

Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: Correlation with DNA‐adduct formation

Ochratoxin A (OTA), a nephrotoxic mycotoxin probably implicated in human Balkan endemic nephropathy and associated urothelial tumors, induces renal carcinomas in rodents and nephrotoxicity in pigs. OTA induces DNA-adduct formation, but the structure of the adducts and their role in nephrotoxicity and carcinogenicity have only partly been elucidated. In vivo, 2-mercaptoethane sulfonate (MESNA) protects rats against OTA-induced nephrotoxicity but not against carcinogenicity, indicating two different mechanisms leading to nephrotoxicity or carcinogenicity. To better understand how DNA-adduct could be generated, opossum kidney cells (OK) have been treated by OTA alone or in presence of several compounds such as MESNA or N-acetylcysteine (another agent that, like MESNA, reduces oxidative stress by increasing of free thiols in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-synthase), and alpha amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (ACIVICIN) (an inhibitor of gamma glutamyl transpeptidase). Cytotoxicity of OTA on OK cells was evaluated by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of the listed agents diminished OTA cytotoxicity significantly; ACIVICIN even increases OTA cytotoxicity. In contrast, analysis of the HPLC profiles of OTA metabolites produced during these incubations indicated that the pattern, the quantity of metabolites, and the nature of the derivatives were modulated by these agents. Ochratoxin B (OTB), open-ring ochratoxin A (OP-OA), 4 hydroxylated OTA, 10 hydroxylated OTA, OTA without phenylalanine, OTB without phenylalanine, and a dechlorinated OTA metabolite could be identified by nano-ESI-IT-MS.