RESUMO
Although celiac disease (CD) is an autoimmune disease that primarily involves the intestinal tract, mounting evidence suggests that a sizeable number of patients exhibit neurological deficits. About 40% of the celiac patients with neurological manifestations have circulating antibodies against neural tissue transglutaminase-6 (tTG6). While early diagnosis and strict adherence to a gluten-free diet (GFD) have been recommended to prevent neurological dysfunction, better therapeutic strategies are needed to improve the overall quality of life. Dysregulation of the microbiota-gut-brain axis, presence of anti-tTG6 antibodies, and epigenetic mechanisms have been implicated in the pathogenesis. It is also possible that circulating or gut-derived extracellular structures and including biomolecular condensates and extracellular vesicles contribute to disease pathogenesis. There are several avenues for shaping the dysregulated gut homeostasis in individuals with CD, non-celiac gluten sensitivity (NCGS) and/or neurodegeneration. In addition to GFD and probiotics, nutraceuticals, such as phyto and synthetic cannabinoids, represent a new approach that could shape the host microbiome towards better prognostic outcomes. Finally, we provide a data-driven rationale for potential future pre-clinical research involving non-human primates (NHPs) to investigate the effect of nutraceuticals, such as phyto and synthetic cannabinoids, either alone or in combination with GFD to prevent/mitigate dietary gluten-induced neurodegeneration.
Assuntos
Canabinoides/uso terapêutico , Doença Celíaca/terapia , Dieta Livre de Glúten/métodos , Disbiose/terapia , Doenças Neurodegenerativas/prevenção & controle , Probióticos/uso terapêutico , Autoanticorpos/sangue , Doença Celíaca/diagnóstico , Doença Celíaca/imunologia , Doença Celíaca/microbiologia , Suplementos Nutricionais , Disbiose/diagnóstico , Disbiose/imunologia , Disbiose/microbiologia , Diagnóstico Precoce , Epigênese Genética , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Glutens/efeitos adversos , Humanos , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/microbiologia , Proteína 2 Glutamina gama-Glutamiltransferase , Qualidade de Vida , Transglutaminases/antagonistas & inibidores , Transglutaminases/genética , Transglutaminases/imunologiaRESUMO
BACKGROUND: Current vector-based malaria control strategies are threatened by the rise of biochemical and behavioural resistance in mosquitoes. Researching mosquito traits of immunity and fertility is required to find potential targets for new vector control strategies. The seminal transglutaminase AgTG3 coagulates male Anopheles gambiae seminal fluids, forming a 'mating plug' that is required for male reproductive success. Inhibitors of AgTG3 can be useful both as chemical probes of A. gambiae reproductive biology and may further the development of new chemosterilants for mosquito population control. METHODS: A targeted library of 3-bromo-4,5-dihydroxoisoxazole inhibitors were synthesized and screened for inhibition of AgTG3 in a fluorescent, plate-based assay. Positive hits were tested for in vitro activity using cross-linking and mass spectrometry, and in vivo efficacy in laboratory mating assays. RESULTS: A targeted chemical library was screened for inhibition of AgTG3 in a fluorescent plate-based assay using its native substrate, plugin. Several inhibitors were identified with IC50 < 10 µM. Preliminary structure-activity relationships within the library support the stereo-specificity and preference for aromatic substituents in the chemical scaffold. Both inhibition of plugin cross-linking and covalent modification of the active site cysteine of AgTG3 were verified. Administration of an AgTG3 inhibitor to A. gambiae males by intrathoracic injection led to a 15% reduction in mating plug transfer in laboratory mating assays. CONCLUSIONS: A targeted screen has identified chemical inhibitors of A. gambiae transglutaminase 3 (AgTG3). The most potent inhibitors are known inhibitors of human transglutaminase 2, suggesting a common binding pose may exist within the active site of both enzymes. Future efforts to develop additional inhibitors will provide chemical tools to address important biological questions regarding the role of the A. gambiae mating plug. A second use for transglutaminase inhibitors exists for the study of haemolymph coagulation and immune responses to wound healing in insects.
Assuntos
Anopheles/enzimologia , Esterilizantes Químicos/farmacologia , Proteínas de Insetos/antagonistas & inibidores , Isoxazóis/farmacologia , Controle de Mosquitos/métodos , Sêmen/enzimologia , Transglutaminases/antagonistas & inibidores , Animais , Domínio Catalítico , Esterilizantes Químicos/síntese química , Esterilizantes Químicos/química , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Isoxazóis/síntese química , Isoxazóis/química , Masculino , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Proteínas Recombinantes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Celiac disease, which mainly affects the small intestine, is the only systemic autoimmune disorder with an identified environmental trigger which is dietary gluten. Lifelong adherence to a strict gluten free diet (GFD) is currently the only accepted treatment. Celiac disease is increasingly diagnosed and the GFD is known to be associated with a large treatment burden. Furthermore, a substantial number of celiac disease patients show an incomplete clinical response to the GFD. These factors have led to demands for the development and testing of novel, non-dietary, therapeutic agents that are both safe and effective. Celiac disease pathogenesis is well elucidated which has greatly aided targeted drug development. Compounds currently being tested in phase II clinical trials include glutenase enzymes (to detoxify gluten) and a tight junction modulator (to reduce access of gluten peptides to lamina propria antigen presenting cells). Other promising approaches include inhibition of the transglutaminase 2 enzyme, blocking antigen presentation by HLA-DQ2 or HLA-DQ8, induction of tolerance, and modulation of the inflammatory response. It is hoped that non-dietary therapy for celiac disease will become available in the coming years and can both reduce the burden of treatment of celiac disease and help patients whose symptoms do not respond completely to the GFD.
Assuntos
Doença Celíaca/terapia , Animais , Anti-Inflamatórios/química , Terapia Biológica/métodos , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Ensaios Clínicos como Assunto , Dieta , Dieta Livre de Glúten , Endopeptidases/química , Proteínas de Ligação ao GTP/antagonistas & inibidores , Glutens/química , Humanos , Camundongos , Oligopeptídeos/química , Peptídeos/química , Proteína 2 Glutamina gama-Glutamiltransferase , Junções Íntimas , Transglutaminases/antagonistas & inibidoresRESUMO
Recently, we reported that Alpinia katsumadai (AK) has anti-nociceptive activity in vivo and that cardamonin (CDN) from AK suppresses the activity and expression of transglutaminase-2 (Tgase-2). However, it remains unknown whether CDN contributes to the anti-nociceptive activities of AK in vivo. We examined the anti-inflammatory effects of CDN in MG63 osteoblast-like cells and Raw264.7 macrophage-like cells treated with interleukin-1ß treatment. CDN suppressed the expression of Tgase-2, cyclooxygenase-2 (COX-2), and p65 (nuclear factor-κB) in a concentration-dependent manner, and restored the expression of IκB in MG63 and Raw264.7 cells. However, CDN did not inhibit the activity of COX-2. Gene silencing of Tgase-2 reduced the COX-2 expression in MG63 cells. Phenylbenzoquinone (PBQ)-induced writhing, carrageenan-induced hyperalgesia, and rota-rod test were used to evaluate the anti-nociceptive activity in vivo. CDN (3-30 mg/kg, orally administered) significantly inhibited PBQ-induced writhing. CDN also produced a significant, dose-dependent increase in the withdrawal response latencies in carrageenan-induced hyperalgesia. The effects of CDN on PBQ-induced writhing were not caused by impaired motor functions. These results suggest that CDN might be helpful in controlling the pain from inflammatory diseases.
Assuntos
Analgésicos/farmacologia , Chalconas/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Transglutaminases/antagonistas & inibidores , Alpinia , Animais , Benzoquinonas/toxicidade , Carragenina/toxicidade , Linhagem Celular , Ciclo-Oxigenase 2/genética , Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Medicina Tradicional Coreana , Camundongos , Camundongos Endogâmicos ICR , Dor/tratamento farmacológico , Plantas Medicinais , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Transglutaminases/genéticaRESUMO
Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Laranja de Acridina , Adenina/análogos & derivados , Adenina/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Cadaverina/análogos & derivados , Caspase 3 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Medicina Tradicional Chinesa , Proteínas de Membrana/biossíntese , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/biossíntese , Transglutaminases/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/biossínteseRESUMO
OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. METHODS AND RESULTS: The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of ß-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit ß-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. CONCLUSIONS: Inhibition of the TG2/ß-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.
Assuntos
Doenças da Aorta/prevenção & controle , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Isoxazóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Quercetina/farmacologia , Transglutaminases/antagonistas & inibidores , Calcificação Vascular/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/induzido quimicamente , Doenças da Aorta/enzimologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Osteogênese/efeitos dos fármacos , Fosforilação , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transglutaminases/genética , Transglutaminases/metabolismo , Calcificação Vascular/induzido quimicamente , Calcificação Vascular/enzimologia , Calcificação Vascular/genética , Calcificação Vascular/patologia , Calcificação Vascular/fisiopatologia , Varfarina , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: We previously demonstrated the efficacy of garlic extract (GE) in the prevention of rat liver fibrosis by inhibiting tissue transglutaminase (tTG) activity. In the present study we aimed to evaluate the potential of GE in the regression of liver fibrosis and the underlining mechanism. METHODS: Male Wistar rats were i.p. injected, twice a week, for 7 weeks, with CCl(4) to develop liver fibrosis. Successively, a group was immediately sacrificed, while the remaining two groups received the GE or the vehicle, respectively, over the following 2 wks. A group of normal rats was also included in the study. Liver function, histology, and collagen deposition in parallel with gene and protein expression of α-SMA, tTG, TGF-ß1, SEMA-7A, and metalloproteinase inhibitor 1 (TIMP1) as well as measure of active by total TGF-ß1 were assessed. RESULTS: CCl(4) administration increased alanine-aminotransferase (ALT) activity, hepatic collagen deposition and gene and protein expression of all monitored markers. GE, but not the sole vehicle, restored liver histology and function by decreasing fibrogenesis markers (α-SMA, tTG, TGF-ß1, SEMA-7A and TIMP1). Active by total TGF-ß1 was significantly reduced (p < 0.05) in GE treated rats compared to the CCl(4) at 7 weeks, and vehicle rats. CONCLUSIONS: These findings concurrently suggested that GE elicited therapeutic effect against liver fibrosis. Regression of liver fibrosis occurred by reducing myofibroblasts (through modulation of HSCs activation mechanisms), remodelling extracellular matrix (through increase of its degradation) and regenerating liver tissue and functions: three processes regulated by fine mechanisms where active TGF-ß1 and tTG play a central role.
Assuntos
Alho/química , Cirrose Hepática/tratamento farmacológico , Extratos Vegetais/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Transglutaminases/antagonistas & inibidores , Actinas/metabolismo , Alanina Transaminase/metabolismo , Animais , Antígenos CD/metabolismo , Tetracloreto de Carbono/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Ratos , Ratos Wistar , Semaforinas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Transglutaminases/metabolismoRESUMO
Tissue transglutaminase (TG2) is a Ca(2+)-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca(2+) activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer's, Huntington's and Parkinson's disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure-activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments.
Assuntos
Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Ligação ao GTP , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Terapia de Alvo Molecular , Proteína 2 Glutamina gama-Glutamiltransferase , Relação Estrutura-AtividadeRESUMO
Inflammatory bowel disease (IBD) represents a socially and clinically relevant disorder, characterized by intestinal chronic inflammation. Cystamine (CysN) is a multipotent molecule with healthy effects and, moreover, it is an inhibitor of transglutaminases (TGs), including the TG type 2 (TG2), an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the IBD. Our aim was to evaluate the effect of CysN in an IBD rat model. A total of 30 rats were divided into 4 groups: controls without treatment (CTR; n=7); receiving the 2,4,6-trinitrobenzene sulfonic acid enema (TNBS group; n=8); treated with TNBS enema plus oral CysN (TNBS-CysN group; n=8); treated with CysN (CysN group; n=7). After killing, bowel inflammation was evaluated applying specific scores. TG activity, TG2 and isopeptide bond immunohistochemical expression, and tumor necrosis factor-α (TNF-α) were evaluated in the colonic tissue, such as interleukin-6 (IL-6) serological levels (ELISA). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared with animal results. TNBS-CysN group developed a less severe colitis compared with the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, P<0.05, respectively) associated with a decrease of TG activity, TG2 and isopeptide bond immunohistochemical expression, TNF-α and IL-6 levels. No statistically significant differences were found between CysN and CTR groups. The colonic immunolocalization of TG2 was comparable in humans affected by IBD and TNBS-administered animals. This is the first demonstration that treatment with a CysN has an anti-inflammatory effect, reducing severity of colitis in a rat model. CysN could be tested as a possible treatment or co-treatment in IBD therapeutic trials.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Colo/efeitos dos fármacos , Cistamina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/prevenção & controle , Transglutaminases/antagonistas & inibidores , Adulto , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Colo/metabolismo , Colo/patologia , Cistamina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-6/sangue , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Transglutaminases/metabolismo , Ácido Trinitrobenzenossulfônico/toxicidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The article by McConoughey et al in the current issue of EMBO Molecular Medicine examines the contribution of transglutaminase 2 (TG2) to Huntington's disease (HD) pathogenesis. The authors find that TG2 inhibition can ameliorate HD neurodegeneration, and thereby elevate the status of transglutaminases (TGs) to a major therapeutic target-not because of their well-known activity in mutant protein aggregation, but instead based upon their ability to epigenetically modulate transcription and energy production. While the reintroduction of TG inhibition as a therapy for HD may evoke feelings of déjà vu, the outcome this time around could go in a dramatically different direction.
Assuntos
Proteínas de Ligação ao GTP/antagonistas & inibidores , Doença de Huntington/enzimologia , Transcrição Gênica , Transglutaminases/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Drosophila , Metabolismo Energético , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Histonas/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Peptídeos/farmacologia , Poliaminas/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética , Transglutaminases/metabolismoRESUMO
BACKGROUND AND AIM: Tissue transglutaminase contributes to liver damage in the development of hepatic fibrosis. In a model of neurodegeneration, the therapeutic benefit of cystamine has been partly attributed to its inhibition of transglutaminase activity. Garlic extract contains many compounds structurally related to cystamine. We investigated the anti-fibrotic effect of garlic extract and cystamine as specific tissue transglutaminase inhibitors. METHODS: Rat liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl(4)) for 7 weeks. Cystamine or garlic extract was administrated by daily intraperitoneal injection, starting from the day after the first administration of CCl(4). Hepatic function, histology, tissue transglutaminase immunostaining and image analysis to quantify Red Sirius stained collagen deposition were examined. Reverse transcription-polymerase chain reaction to detect alpha-SMA, IL-1beta and tissue transglutaminase expression and Western blot for tissue transglutaminase protein amount were performed. Transglutaminase activity was assayed on liver homogenates by a radio-enzymatic method. RESULTS: Transglutaminase activity was increased in CCl(4) group and reduced by cystamine and garlic extract (p<0.05). Treatment with cystamine and garlic extract reduced the liver fibrosis and collagen deposition, particularly in the garlic extract group (p<0.01). Moreover, the liver damage improved and serum alanine aminotransferase was decreased (p<0.05). Tissue transglutaminase immunolocalised with collagen fibres and is mainly found in the ECM of damaged liver. Alpha-SMA, IL-1beta, tissue transglutaminase mRNA and tissue transglutaminase protein were down-regulated in the cystamine and garlic extract groups compared to controls. CONCLUSION: These findings concurrently suggest that transglutaminase may play a pivotal role in the pathogenesis of liver fibrosis and may identify garlic cystamine-like molecules as a potential therapeutic strategy in the treatment of liver injury.
Assuntos
Cistamina/administração & dosagem , Alho , Cirrose Hepática/enzimologia , Cirrose Hepática/terapia , Extratos Vegetais/administração & dosagem , Transglutaminases/sangue , Actinas/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Colágeno/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Inibidores Enzimáticos/administração & dosagem , Injeções Intraperitoneais , Interleucina-1beta/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Testes de Função Hepática , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transglutaminases/antagonistas & inibidoresRESUMO
Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the posttranslational modification of intra- and extracellular proteins and implicated in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (LOPAC and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor, and its metabolite ZM449829 were the most potent inhibitors with IC(50) of 10 and 5 nM, respectively. In addition, two other inhibitors, including tyrphostin 47 and vitamin K(3), were found to have an IC(50) in the micromolar range. These agents used in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intramolecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Animais , Cálcio/farmacologia , Técnicas de Química Combinatória , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Avaliação Pré-Clínica de Medicamentos , Fator XIIIa/antagonistas & inibidores , Fator XIIIa/metabolismo , Proteínas de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Humanos , Doença de Machado-Joseph/enzimologia , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Octoxinol , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/metabolismo , Tirfostinas/químicaRESUMO
Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 degrees C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.
Assuntos
Cistamina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Traumatismo por Reperfusão/enzimologia , Transglutaminases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Temperatura Baixa , Cistamina/análise , Inibidores Enzimáticos/análise , L-Lactato Desidrogenase , Miócitos Cardíacos/enzimologia , Soluções para Preservação de Órgãos/química , Poliaminas/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Ratos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Transglutaminases/antagonistas & inibidores , Caseínas/química , Compostos de Dansil/química , Lisina/análogos & derivados , Lisina/química , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
Herein we report the synthesis of 24 novel peptides as potential irreversible inactivators of transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include either alpha,beta-unsaturated amide groups or the corresponding epoxide groups. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Amidas/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Compostos de Epóxi/química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fígado/enzimologia , Transglutaminases/antagonistas & inibidores , Animais , Bioquímica/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Cobaias , Cinética , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Herein, we report the results of irreversible inhibition of guinea pig liver transglutaminase (TGase) by a series of 24 novel dipeptides containing either an alpha,beta-unsaturated amide or an epoxide functional group. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 421 x 10(3) and 3000 x 10(3)M(-1)min(-1).
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Fígado/efeitos dos fármacos , Transglutaminases/antagonistas & inibidores , Amidas/química , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Compostos de Epóxi/química , Cobaias , Cinética , Fígado/enzimologia , Espectroscopia de Ressonância MagnéticaRESUMO
Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both mu- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.
Assuntos
Baculoviridae/genética , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Transglutaminases/química , Animais , Cálcio/metabolismo , Calpaína/metabolismo , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Guanosina Trifosfato/farmacologia , Humanos , Hidrólise , Óxido Nítrico/farmacologia , Fosforilcolina/farmacologia , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Esfingosina/farmacologia , Spodoptera , Transglutaminases/antagonistas & inibidores , Transglutaminases/isolamento & purificação , Transglutaminases/metabolismoRESUMO
TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.
Assuntos
Macrófagos Peritoneais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Northern Blotting , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Linhagem Celular , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hidrocortisona/farmacologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Fator de Crescimento Insulin-Like II/farmacologia , Lipopolissacarídeos , Macrófagos Peritoneais/efeitos dos fármacos , Manosefosfatos/farmacologia , Camundongos , Reação em Cadeia da Polimerase , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/antagonistas & inibidores , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.