RESUMO
Pt(ii)-based antitumor drugs (e.g. cisplatin and oxaliplatin) are one of the most successful and frequently used drugs in cancer chemotherapy at present. However, drug resistance and severe side effects are the major problems in the application of platinum drugs. Detoxification of Pt(ii) drugs is one of the most important mechanisms of drug resistance. Herein, a supramolecular Pt(iv) prodrug nano-assembly delivery system is designed and used to encapsulate a γ-glutamyl transferase (GGT) inhibitor (OU749) (Pt-CD/Dex-Ad@OU nano-assemblies) for the synergistic chemotherapy of cisplatin-resistant cancer. Pt-CD/Dex-Ad@OU nano-assemblies could be efficiently taken up by cisplatin-resistant cancer cells and release a drug in the intracellular reductive environment. The Pt-CD/Dex-Ad@OU nano-assemblies can efficiently suppress the expression of GGT, depleting GSH and augmenting ROS via the reduction of the Pt(iv) prodrug. Thereby, by breaking the redox balance the detoxification and antiapoptosis mechanisms of Pt(ii) drugs can be overcome. Thereafter, the excellent therapeutic efficacy of Pt-CD/Dex-Ad@OU nano-assemblies is validated on a cisplatin-resistant human non-small cell lung cancer (A549/DDP) model. Furthermore, the inhibition of GGT protein is expected to reduce the nephrotoxicity of cisplatin. Collectively, this study provides a promising strategy to break the redox balance for overcoming drug resistance and maximizing the efficacy of platinum-based cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pró-Fármacos/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , HumanosRESUMO
AIM: Cisplatin is a potent chemotherapeutic agent whose therapeutic application is hindered by the associated nephrotoxicity. Cisplatin-evoked nephrotoxicity has been largely attributed to the induction of oxidative stress and inflammatory responses. The current study aimed at investigating the ability of ergothioneine to mitigate cisplatin-evoked nephrotoxicity and to elucidate the underlining molecular mechanisms. MAIN METHODS: Wistar rats were treated with a daily dose of ergothioneine (70 mg/kg, po) for fourteen days and a single dose of cisplatin (5 mg/kg, ip) on day ten. On day fifteen, kidneys and blood specimens were collected and subjected to Western blotting, ELISA, histopathological, and spectrophotometric analysis. KEY FINDINGS: Ergothioneine significantly enhanced renal function in cisplatin-treated rats as manifested by increased GFR and decreased serum creatinine and blood urea nitrogen. Ergothioneine effectively reduced the cisplatin-induced oxidative stress and mitigated apoptosis and the histopathological changes. Mechanistically, ergothioneine induced the expression of the antioxidant transcription factor Nrf2 and up-regulated its downstream targets NQO1 and HO-1. Equally important, ergothioneine inhibited γ-glutamyl transpeptidase that plays crucial roles in biotransformation of cisplatin into a toxic metabolite. Additionally, it reduced the pro-apoptotic protein p53 and the inflammatory transcription factor NF-κB along with its downstream pro-inflammatory cytokines TNF-α and IL-1ß. SIGNIFICANCE: The results of the current work shed the light on the ameliorating effect of ergothioneine on cisplatin-evoked nephrotoxicity that is potentially mediated through modulation of Nrf2, p53, and NF-κB signaling and inhibition of γ-glutamyl transpeptidase. This findings support the potential application of ergothioneine in controlling cisplatin-associated nephrotoxicity although clinical investigations are warranted.
Assuntos
Cisplatino/farmacologia , Ergotioneína/farmacologia , Rim/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose , Caspase 3/metabolismo , Fragmentação do DNA , Masculino , Estresse Oxidativo , Ratos , Ratos Wistar , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , gama-Glutamiltransferase/metabolismoRESUMO
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.
Assuntos
Dipeptídeos/metabolismo , Inibidores Enzimáticos/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dipeptídeos/química , Humanos , Modelos Moleculares , Conformação Proteica , gama-Glutamiltransferase/química , gama-Glutamiltransferase/metabolismoRESUMO
AIMS: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. RESULTS: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. CONCLUSION: s. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.
Assuntos
Anetol Tritiona/administração & dosagem , Glutationa/metabolismo , gama-Glutamiltransferase/metabolismo , Anetol Tritiona/farmacologia , Animais , Linhagem Celular , Cisteína/sangue , Cisteína/metabolismo , Dipeptídeos/sangue , Dipeptídeos/metabolismo , Dissulfetos/sangue , Glutationa/sangue , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Malondialdeído/sangue , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
Gamma-glutamyltransferase 5 (GGT5) is a member of the gamma-glutamyl transpeptidase gene family with the capacity of cleaving the gamma-glutamyl moiety of glutathione, but its role in cancer progression has never been revealed. In this study, we found that gene GGT5 was highly expressed in cancer-associated fibroblasts (CAFs) in lung adenocarcinoma, predicting the unfavorable survival of patients with lung adenocarcinoma. Cell growth, foci formation and spheres formation analyses showed that cancer cell proliferation was attenuated under treatment with the conditioned media from GGT5-silenced CAFs. Moreover, high expression of GGT5 in CAFs enhanced the drug resistance of cancer cells by increasing intracellular glutathione and reducing the intracellular reactive oxygen species in cancer cells. In mouse xenograft model, we proved that targeting GGT5 with a small-molecule inhibitor GGsTop could inhibit tumor growth and increase the chemosensitivity of cancer cells. Taken together, our study illuminates that high level of GGT5 in CAFs contributes to cancer cell survival and drug resistance, indicating that GGT5 may be a promising therapeutic target in lung adenocarcinoma.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Fibroblastos Associados a Câncer/patologia , Neoplasias Pulmonares/tratamento farmacológico , gama-Glutamiltransferase/metabolismo , Células A549 , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fibroblastos Associados a Câncer/metabolismo , Proliferação de Células/efeitos dos fármacos , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Humanos , Estimativa de Kaplan-Meier , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.
Assuntos
Aminobutiratos/farmacologia , Isquemia Miocárdica/fisiopatologia , Organofosfonatos/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/fisiologia , Animais , Coração/fisiopatologia , Masculino , Malondialdeído/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Norepinefrina/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , gama-Glutamiltransferase/metabolismoRESUMO
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.
Assuntos
Organismos Aquáticos/química , Histidina/farmacologia , Compostos de Enxofre/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , Compostos Azo/farmacologia , Proliferação de Células/efeitos dos fármacos , Desenvolvimento de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios Enzimáticos , Glutationa/metabolismo , Células HEK293 , Histidina/química , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Norleucina/análogos & derivados , Norleucina/farmacologia , Especificidade por Substrato , Compostos de Enxofre/química , Testes de Toxicidade , gama-Glutamiltransferase/metabolismoRESUMO
γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 µm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.
Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Metilistidinas/farmacologia , gama-Glutamiltransferase/genética , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glutationa/genética , Histidina/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Oxirredução , Proteólise , Especificidade por Substrato , Compostos de Enxofre/farmacologia , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
AIMS: Soluble dipeptidyl peptidase-4 (sDPP-4) is secreted by hepatocytes and induces adipose tissue inflammation and insulin resistance. Sodium-glucose co-transporter-2 (SGLT2) inhibitors can improve hepatic steatosis by inhibiting hepatic de novo lipogenesis. We investigated the effects of dapagliflozin (an SGLT2 inhibitor) on serum levels of sDPP-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-seven patients with type 2 diabetes and NAFLD were randomized to a dapagliflozin group (5 mg/d for 24 weeks) (n = 33) or the control group (n = 24). Serum levels of sDPP-4 were measured with a commercial ELISA kit. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured by dual bioelectrical impedance analysis. RESULTS: In a total of 57 patients, baseline serum sDPP-4 was positively correlated with aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT) and HOMA-IR Both VAT and SAT areas decreased significantly in the dapagliflozin group alone. Liver enzymes were decreased at 24 weeks in the dapagliflozin group, but were unchanged in the control group. Although both groups showed significant reduction of serum sDPP-4 after 24 weeks of treatment, the magnitude of decrease was significantly larger in the dapagliflozin group. Changes in liver enzymes during treatment with dapagliflozin were positively correlated with the change in serum sDPP-4, but not with changes in VAT volume or HbA1c. CONCLUSIONS: Improvement of liver dysfunction after treatment with dapagliflozin was associated with a decrease in serum sDPP-4, suggesting that reduction of serum sDPP-4 by SGLT2 inhibitors may be a therapeutic strategy for NAFLD/NASH in patients with type 2 diabetes that is independent of glucose lowering or weight loss.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Compostos Benzidrílicos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Dipeptidil Peptidase 4/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Feminino , Glucosídeos , Hepatite/complicações , Humanos , Inflamação/complicações , Resistência à Insulina/fisiologia , Gordura Intra-Abdominal/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Gordura Subcutânea/efeitos dos fármacos , Redução de Peso/fisiologia , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: GGsTop treated OM quickly and safely. GGsTop is highly valuable for use as a treatment for OM.
Assuntos
Aminobutiratos/administração & dosagem , Fluoruracila/efeitos adversos , Organofosfonatos/administração & dosagem , Estomatite/tratamento farmacológico , gama-Glutamiltransferase/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Fluoruracila/administração & dosagem , Humanos , Camundongos , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Estomatite/induzido quimicamente , Estomatite/genética , Estomatite/patologia , gama-Glutamiltransferase/genéticaRESUMO
Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.
Assuntos
Fase de Clivagem do Zigoto/efeitos dos fármacos , Glutationa Sintase/metabolismo , Glutationa/farmacologia , Zigoto/efeitos dos fármacos , gama-Glutamiltransferase/metabolismo , Animais , Bovinos , Fase de Clivagem do Zigoto/metabolismo , Técnicas de Cultura Embrionária , Inibidores Enzimáticos/farmacologia , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Glutationa/metabolismo , Glutationa Sintase/antagonistas & inibidores , Glutationa Sintase/genética , Masculino , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Zigoto/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are secondary plant ingredients formed in many plant species to protect against predators. PAs are generally considered acutely hepatotoxic, genotoxic and carcinogenic. Up to now, only few in vitro and in vivo investigations were performed to evaluate their relative toxic potential. AIM OF THE STUDY: The aim was to develop an in vitro screening method of their cytotoxicity. MATERIALS AND METHODS: Human and rodent hepatocyte cell lines (HepG2 and H-4-II-E) were used to assess cytotoxicity of the PA lasiocarpine. At concentrations of 25⯵M up to even 2400⯵M, no toxic effects in neither cell line was observed with standard cell culture media. Therefore, different approaches were investigated to enhance the susceptibility of cells to PA toxicity (using high-glucose or galactose-based media, induction of toxifying cytochromes, inhibition of metabolic carboxylesterases, and inhibition of glutathione-mediated detoxification). RESULTS: Galactose-based culture medium (11.1â¯mM) increased cell susceptibility in both cell-lines. Cytochrome P450-induction by rifampicin showed no effect. Inhibition of carboxylesterase-mediated PA detoxification by specific carboxylesterase 2 inhibitor loperamide (2.5⯵M) enhanced lasiocarpine toxicity, whereas the unspecific carboxylesterase inhibitor bis(4-nitrophenyl)phosphate (BNPP, 100⯵M)) had a weaker effect. Finally, the inhibition of glutathione-mediated detoxification by buthionine sulphoximine (BSO, 100⯵M) strongly enhanced lasiocarpine toxicity in H-4-II-E cells in low and medium, but not in high concentrations. CONCLUSIONS: If no toxicity is observed under standard conditions, susceptibility enhancement by using galactose-based media, loperamide, and BSO may be useful to assess relative acute cytotoxicity of PAs in different cell lines.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Alcaloides de Pirrolizidina/toxicidade , Testes de Toxicidade Aguda , Ativação Metabólica , Animais , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Meios de Cultura/metabolismo , Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Fígado/enzimologia , Fígado/patologia , Alcaloides de Pirrolizidina/metabolismo , Ratos , Medição de Risco , Fatores de Tempo , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/metabolismoRESUMO
The Notch pathway has been reported to control tissue damage in acute kidney diseases. To investigate potential beneficial nephroprotective effects of targeting Notch, we developed chemically functionalized γ-secretase inhibitors (GSIs) targeting γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT), two enzymes overexpressed in the injured kidney, and evaluated them in in vivo murine models of acute tubular and glomerular damage. Exposure of the animals to disease-inducing drugs together with the functionalized GSIs improved proteinuria and, to some extent, kidney dysfunction. The expression of genes involved in the Notch pathway, acute inflammatory stress responses, and the renin-angiotensin system was enhanced in injured kidneys, which could be downregulated upon administration of functionalized GSIs. Immunohistochemistry staining and Western blots demonstrated enhanced activation of Notch1 as detected by its cleaved active intracellular domain during acute kidney injury, and this was downregulated by concomitant treatment with the functionalized GSIs. Thus targeted γ-secretase-based prodrugs developed as substrates for γ-GT/γ-GCT have the potential to selectively control Notch activation in kidney diseases with subsequent regulation of the inflammatory stress response and the renin-angiotensin pathways.
Assuntos
Injúria Renal Aguda/prevenção & controle , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Rim/efeitos dos fármacos , Receptor Notch1/metabolismo , gama-Glutamilciclotransferase/antagonistas & inibidores , gama-Glutamiltransferase/antagonistas & inibidores , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Citoproteção , Modelos Animais de Doenças , Rim/enzimologia , Rim/patologia , Masculino , Camundongos Endogâmicos BALB C , Proteinúria/enzimologia , Proteinúria/patologia , Proteinúria/prevenção & controle , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismoRESUMO
2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.
Assuntos
Aminobutiratos/síntese química , Inibidores Enzimáticos/síntese química , Organofosfonatos/síntese química , gama-Glutamiltransferase/antagonistas & inibidores , Aminobutiratos/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Organofosfonatos/metabolismo , Ligação Proteica , Estereoisomerismo , gama-Glutamiltransferase/metabolismoRESUMO
We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Compostos de Espiro/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , Células A549 , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Espécies Reativas de Oxigênio/metabolismo , Compostos de Espiro/química , gama-Glutamiltransferase/metabolismoRESUMO
γ-Glutamyl transpeptidase (GGT) is a cell-membrane-bound enzyme that is involved in various physiological and pathological processes and is regarded as a potential biomarker for many malignant tumors, precise detection of which is useful for early cancer diagnosis. Herein, a new GGT-activatable near-infrared (NIR) fluorescence imaging probe (GANP) by linking of a GGT-recognitive substrate γ-glutamate (γ-Glu) and a NIR merocyanine fluorophore (mCy-Cl) with a self-immolative linker p-aminobenzyl alcohol (PABA) is reported. GANP was stable under physiological conditions, but could be efficiently activated by GGT to generate ≈100-fold enhanced fluorescence, enabling high sensitivity (detection limit of ≈3.6 mU L-1 ) and specificity for the real-time imaging of GGT activity as well as rapid evaluation of the inhibition efficacy of GGT inhibitors in living tumor cells. Notably, the deep tissue penetration ability of NIR fluorescence could further allow GANP to image GGT in frozen tumor tissue slices with large penetration depth (>100â µm) and in xenograft tumors in living mice. This GGT activatable NIR fluorescence imaging probe could facilitate the study and diagnosis of other GGT-correlated diseases in vivo.
Assuntos
Corantes Fluorescentes/metabolismo , Neoplasias/patologia , gama-Glutamiltransferase/metabolismo , Animais , Benzopiranos/química , Álcoois Benzílicos/química , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Células HCT116 , Humanos , Indóis/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Espectroscopia de Luz Próxima ao Infravermelho , Transplante Heterólogo , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM-1 min-1 and the Ki was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.
Assuntos
Diazo-Oxo-Norleucina/química , Inibidores Enzimáticos/química , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Estrutura Secundária de ProteínaRESUMO
γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.
Assuntos
Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Lisina/antagonistas & inibidores , Organofosfonatos/farmacologia , gama-Glutamiltransferase/antagonistas & inibidores , Domínio Catalítico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lisina/metabolismo , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/síntese química , Organofosfonatos/química , Eletricidade Estática , Relação Estrutura-Atividade , gama-Glutamiltransferase/química , gama-Glutamiltransferase/metabolismoRESUMO
The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity inâ vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for inâ vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3â ppm) and the T1 of both compounds is relatively long (30â s and 45â s, respectively, in H2 O at 9.4â T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to inâ vivo modulation of GGT activity induced by the inhibitor acivicin.
Assuntos
Ensaios Enzimáticos/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , gama-Glutamiltransferase/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Isoxazóis/farmacologia , Sondas Moleculares/metabolismo , Ratos , gama-Glutamiltransferase/antagonistas & inibidoresRESUMO
Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible γ-glutamyl transpeptidase (γ-GT) inhibitor. We studied the protective effects of GGsTop on IR-induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body wt of GGsTop (IR-GGsTop group) was injected into the liver through the inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow and at 12, 24, and 48 h after reperfusion. All the animals were euthanized at 48 h after reperfusion and the livers were harvested. Serum levels of alanine transaminase, aspartate transaminase, and γ-GT were significantly lower after reperfusion in the IR-GGsTop group compared with the IR group. Massive hepatic necrosis was present in the IR group, while only few necroses were present in the IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content, which was significantly reduced in the IR group. Furthermore, GGsTop prevented increase of hepatic γ-GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that led to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events.