RESUMO
BACKGROUND: NASH causes a tremendous health care burden in the United States. A glucagon-like peptide-1 agonist, semaglutide (Sema), treatment resulted in hepatic steatosis reduction in clinical trials of NASH. Lysophosphatidic acid receptor 1 antagonists are known to have antifibrotic effects in several organs. We tested Sema and a novel lysophosphatidic acid receptor 1 antagonist, EPGN2154, individually and in combination to evaluate their efficacy for NASH remission in preclinical models. METHODS: In the present study, we used (1) C57Bl6/J wild-type mice fed on a high-fat, high-carbohydrate (HFHC) diet for 16 weeks and (2) leptin-deficient mice (ob/ob) fed on an Amylin liver NASH diet for 16 weeks. After 16 weeks, the mice were randomly distributed in equal numbers in (1) no-drug, (2) EPGN2154, (3) Sema, and (4) EPGN2154+Sema treatment groups for 8 additional weeks at a dosage of 10 mg/kg body weight for EPGN2154 (oral gavage, 5 days a week) and 6.17 µg/kg body weight of Sema (subcutaneous injection every alternate day, 3 days a week). RESULTS: In the wild-type-high-fat, high-carbohydrate model, we observed the most body weight loss in the EPGN2154+Sema combination group compared to the other treatment groups. All groups led to a significant reduction in alanine transaminase levels when compared to high-fat, high-carbohydrate-fed wild type. However, no significant difference in alanine transaminase levels was observed among the treatment groups. In the ob/ob mice study, Sema did not cause body weight loss. Moreover, the EPGN2154 and the combination groups had a lower NAFLD Activity Score and incidence of advanced-stage hepatic fibrosis than the Sema group. CONCLUSIONS: EPGN2154 demonstrated a hepato-protective effect independent of body weight loss in preclinical NASH models.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores de Ácidos Lisofosfatídicos/uso terapêutico , Alanina Transaminase , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Carboidratos/uso terapêutico , Redução de PesoRESUMO
Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-ß pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática/genética , MAP Quinase Quinase Quinases/metabolismo , Miofibroblastos/metabolismo , Animais , Biópsia , Diferenciação Celular , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Camundongos , Camundongos SCID , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tomografia Computadorizada por Raios X , Fator de Crescimento Transformador beta/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients.
Assuntos
Mediadores da Inflamação/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Pneumonia/enzimologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Sistema Respiratório/enzimologia , Fumar/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Linhagem Celular Tumoral , Citocinas/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/genética , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Pneumonia/etiologia , Pneumonia/genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Interferência de RNA , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-ß has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-ß-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-ß stimulation. TGF-ß-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.
Assuntos
Bleomicina/farmacologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Lavagem Broncoalveolar/métodos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Células HeLa , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Indóis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Piridonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.
Assuntos
Plaquetas/citologia , Diferenciação Celular/fisiologia , Megacariócitos/citologia , Megacariócitos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Southern Blotting , Western Blotting , Células da Medula Óssea/citologia , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Receptores de Lisoesfingolipídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombopoetina/sangueRESUMO
Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D(2). We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD(2) receptor; chemoattractant receptor-homologous molecule expressed on T(h) class 2 cells (CRT(h)2). Activation of CRT(h)2 on BMMC by PGD(2) or the CRT(h)2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D(2) (DK-PGD(2)), resulted in signaling response including Ca(2+) mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT(h)2, Compound A. Activation of CRT(h)2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD(2) and DK-PGD(2) induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD(2) injection. Both these processes were inhibited by the CRT(h)2 antagonist. These results raise the possibility that the functional consequences of the PGD(2)-CRT(h)2 interaction on mast cells may be relevant in allergic inflammation.
Assuntos
Mastócitos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Medula Óssea/patologia , Cálcio/imunologia , Cálcio/metabolismo , Movimento Celular/imunologia , Células Cultivadas , Hipersensibilidade Tardia/imunologia , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismo , Selectina L/genética , Selectina L/metabolismo , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Transdução de SinaisRESUMO
A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.
Assuntos
Dermatite Alérgica de Contato/tratamento farmacológico , Antagonistas de Prostaglandina/uso terapêutico , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Linhagem Celular , Citocinas/imunologia , Dermatite Alérgica de Contato/imunologia , Feminino , Fluoresceína-5-Isotiocianato/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina D2/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Prostaglandin D(2) (PGD(2)) and its receptor chemoattractant receptor homologous molecule expressed on T(h)2 cells (CRTH2) have been implicated in the pathogenesis of numerous allergic diseases. We investigated the role of PGD(2) and CRTH2 in allergic cutaneous inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated cutaneous inflammation caused by either repeated epicutaneous ovalbumin or FITC sensitization. Gene expression and ELISA analysis revealed that there was reduced pro-inflammatory cytokine mRNA or protein produced. Importantly, the CRTH2 antagonist reduced total IgE, as well as antigen-specific IgE, IgG1 and IgG2a antibody levels. This reduction in antibody production correlated to reduced cytokines produced by splenocytes following in vitro antigen challenge. An examination of skin CD11c(+) dendritic cells (DC) showed that in mice treated with the CRTH2 antagonist, there was a decrease in the number of these cells that migrated to the draining lymph nodes in response to FITC application to the skin. Additionally, naive CD4(+) T lymphocytes co-cultured with skin-derived DC from CRTH2 antagonist-treated mice showed a reduced ability to produce a number of cytokines compared with DC from vehicle-treated mice. Collectively, these findings suggest that CRTH2 has a pivotal role in mediating the inflammation and the underlying immune response following epicutaneous sensitization.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/análise , Células Dendríticas/imunologia , Dermatite Atópica/imunologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Imunoglobulinas/sangue , Imunoglobulinas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Antagonistas de Prostaglandina/farmacologia , Prostaglandina D2/imunologia , Prostaglandina D2/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Regulação para CimaRESUMO
Prostaglandin D(2), the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD(2) and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD(2)-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation.
Assuntos
Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/prevenção & controle , Regulação para Baixo/genética , Pneumonia/complicações , Pneumonia/genética , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Alérgenos/imunologia , Animais , Anticorpos/sangue , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/imunologia , Baratas , Citocinas/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/sangue , Pneumonia/imunologiaRESUMO
The alpha4beta1 integrin (very late antigen-4, VLA-4) plays an important role in the migration of lymphocytes, monocytes, and eosinophils, but not neutrophils, to sites of inflammation. Pharmacological antagonism of VLA-4 is an attractive prospect for the treatment of predominantly eosinophil mediated diseases such as asthma and allergic rhinitis. We report here on a potent and selective, small molecule VLA-4 inhibitor, (2S)-3-(2', 5'-dichlorobiphenyl-4-yl)-2-({[1-(2-methoxybenzoyl)piperidin-3-yl]carbonyl}amino) propanoic acid, compound 1, and characterize the antagonist activities of this molecule in various cell-based assays and in an animal model of eosinophil migration. Compound 1 inhibited VLA-4/ vascular cell adhesion molecule-1(VCAM-1) interactions with in vitro potencies (IC50 value of 210 nM) in VLA-4-expressing Ramos cells, although the compound did not inhibit cell adhesion to fibronectin via alpha5beta1 integrin (very late antigen-5, VLA-5). Blockade of phorbol-12-myristate-13-acetate (PMA)- or Mn2+-stimulated VLA-4 interactions with compound 1 was observed in human T lymphocytes (IC50 value of 230 nM), human eosinophils (IC50 value of 4.0 microM) and mouse eosinophils (IC50 value of 1.6 microM). Furthermore, compound 1 administered by intraperitoneal injection inhibited eosinophil infiltration in a dose-dependent manner by up to 80% in an air pouch model. These data support the use of small molecule VLA-4 antagonists in the treatment of relevant diseases, such as asthma, atopic dermatitis, or allergic rhinitis.
Assuntos
Antialérgicos/farmacologia , Anti-Inflamatórios/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinofilia/prevenção & controle , Eosinófilos/efeitos dos fármacos , Integrina alfa4beta1/antagonistas & inibidores , Bifenilos Policlorados/farmacologia , Dermatopatias/prevenção & controle , Animais , Antialérgicos/farmacocinética , Antialérgicos/uso terapêutico , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Adesão Celular/efeitos dos fármacos , Quimiocina CCL11 , Quimiocinas CC , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eosinofilia/induzido quimicamente , Eosinofilia/metabolismo , Eosinofilia/fisiopatologia , Eosinófilos/metabolismo , Feminino , Fibronectinas/metabolismo , Humanos , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/metabolismo , Interleucina-5/biossíntese , Interleucina-5/genética , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Bifenilos Policlorados/farmacocinética , Bifenilos Policlorados/uso terapêutico , Dermatopatias/induzido quimicamente , Dermatopatias/metabolismo , Dermatopatias/fisiopatologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The cinnabaramides A-G (1-7) were isolated from a terrestrial strain of Streptomyces as potent and selective inhibitors of the human 20S proteasome. Their chemical and biological properties resemble those of salinosporamide A, a recently identified lead compound from an obligate marine actinomycete, which is currently under development as an anticancer agent. Cinnabaramides F and G (6, 7) combine essential structural features of salinosporamide A and lactacystin and show about equal potency in vitro, with IC50 values in the 1 nM range. The properties and phylogenetic position of the producer organism, the production and isolation of compounds 1-7, their structure elucidation by MS and NMR, and their biological activities are reported. Additionally, an X-ray crystal structure was obtained from cinnabaramide A (1).
Assuntos
Acetilcisteína/análogos & derivados , Lactonas , Inibidores de Proteassoma , Pirróis , Streptomyces/química , Acetilcisteína/sangue , Acetilcisteína/química , Acetilcisteína/isolamento & purificação , Acetilcisteína/farmacologia , Cristalografia por Raios X , Humanos , Lactonas/sangue , Lactonas/isolamento & purificação , Lactonas/farmacologia , Conformação Molecular , Estrutura Molecular , Pirróis/sangue , Pirróis/isolamento & purificação , Pirróis/farmacologiaRESUMO
BACKGROUND: To investigate the functional role of protease activated receptor (PAR) -2 in T lymphocytes, we analyzed TCR-mediated inflammatory cytokine production using PAR-2 deficient (KO) and wild type (WT) mice. METHODS: Production of serum IgE and cytokines by spleen CD4+ T cells was determined in OVA-sensitized and OVA-challenged mice of PAR-2 KO in contrast to WT mice. Phosphorylation of JNK1 and 2 was determined by Western blotting. RESULTS: A reduction in serum levels of IgE and IL-4 production by splenic CD4+ T cells from OVA-sensitized and OVA-challenged KO mice compared to WT mice was observed. By contrast, IFN-gamma production was upregulated after antigen stimulation in KO mice. Anti-CD3-mediated phosphorylation of JNK1 was upregulated in splenic CD4+ T cells from KO mice compared to WT mice. CONCLUSIONS: PAR-2 participates in the regulation of T cell cytokine production that may be caused by modulation of JNK1 phosphorylation.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Regulação para Baixo/genética , Interferon gama/biossíntese , Interleucina-4/antagonistas & inibidores , Receptor PAR-2/deficiência , Regulação para Cima/genética , Animais , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Regulação para Baixo/imunologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Ovalbumina/imunologia , Fosforilação , Receptor PAR-2/genética , Regulação para Cima/imunologiaRESUMO
We previously showed that ramatroban (Baynastrade mark), a thromboxane A(2) (TxA(2)) antagonist, had inhibited prostaglandin D(2) (PGD(2))-stimulated human eosinophil migration mediated through activation of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, detailed pharmacological characterization of its inhibitory activity has not been described. In the present study, we showed that [(3)H]ramatroban bound to a single receptor site on CRTH2 transfectants with a similar K(d) value (7.2 nM) to a TxA(2) receptor (8.7 nM). We also demonstrated that ramatroban inhibited PGD(2)-, 15-deoxy-Delta(12, 14)-PGJ(2) (15d-PGJ(2))- and indomethacin-induced calcium responses on CRTH2 transfectants in a competitive manner with similar pA(2) values (8.5, 8.5, and 8.6, respectively). This is the first report showing the evidence for direct binding of ramatroban to CRTH2, revealing its competitive inhibitory effects and another interesting finding that PGD(2), indomethacin and 15d-PGJ(2) share the same binding site with ramatroban on CRTH2.
Assuntos
Carbazóis/farmacologia , Indometacina/farmacologia , Prostaglandina D2/farmacologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Sulfonamidas/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes , Cálcio/metabolismo , Carbazóis/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados , Humanos , Hidrazinas/farmacologia , Modelos Biológicos , Ácidos Pentanoicos/farmacologia , Prostaglandina D2/análogos & derivados , Piridinas/farmacologia , Receptores Imunológicos/agonistas , Receptores Imunológicos/genética , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/genética , Sulfonamidas/metabolismo , Transfecção , TrítioRESUMO
The discovery of a second receptor for prostaglandin D2, chemoattractant receptor-homologous molecule expressed on T helper type 2 (CRTH2), has revived significant efforts into the development of small-molecule antagonists for allergic diseases as the receptor is predominantly expressed on cells such as eosinophils, TH2 cells and basophils, which are major pro-inflammatory cells in diseases such as asthma and allergic rhinitis. This brief review serves to illustrate current patent literature focusing on the similarities and differences of an ever-growing number of CRTH2 antagonists emerging from corporate laboratories.
Assuntos
Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/patologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/fisiologia , Antagonistas de Prostaglandina/uso terapêutico , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/fisiologia , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/fisiologia , Animais , Humanos , Hipersensibilidade/imunologia , Antagonistas de Prostaglandina/farmacologiaRESUMO
1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell-mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF-kappaB). 3 NF-kappaB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK-beta. 4 We identified COMPOUND A as a small-molecule, ATP-competitive inhibitor selectively targeting IKK-beta kinase activity with a K(i) value of 2 nM. 5 COMPOUND A inhibited stress-induced NF-kappaB transactivation, chemokine-, cytokine-, and adhesion molecule expression, and T- and B-cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS-induced TNF-alpha in rodents. 7 In mice COMPOUND A inhibited cockroach allergen-induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed-type hypersensitivity. 10 These data suggest that IKK-beta inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Oxazinas/farmacologia , Pneumonia/prevenção & controle , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/farmacocinética , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/biossíntese , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Edema/prevenção & controle , Feminino , Humanos , Quinase I-kappa B , Proteínas I-kappa B/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Oxazinas/efeitos adversos , Oxazinas/farmacocinética , Fosforilação , Pneumonia/imunologia , Piridinas/efeitos adversos , Piridinas/farmacocinética , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.
Assuntos
Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/farmacologia , Mieloma Múltiplo/patologia , NF-kappa B/farmacologia , Ácidos Nicotínicos/farmacologia , Nitrilas/farmacologia , Proliferação de Células , Citocinas/biossíntese , DNA/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Fosforilação , Células Tumorais Cultivadas , Regulação para CimaRESUMO
A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as IkappaB kinase beta (IKK-beta) inhibitors. Substitution of an aminoalkyl group for the aromatic group at the 4-position on the core pyridine ring resulted in a marked increase in both kinase enzyme and cellular potencies, and provided potent IKK-beta inhibitors with IC(50) values of below 100 nM.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Quinase I-kappa B , Cinética , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as I kappaB kinase beta (IKK-beta) inhibitors. Modification of a novel IKK-beta inhibitor 1 (IKK-beta IC(50)=1500 nM, Cell IC(50)=8000 nM) at the 4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro profiles (IKK-beta IC(50)=8.5 nM, Cell IC(50)=60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects at 1mg/kg, po in arachidonic acid-induced ear edema model in mice).
Assuntos
Anti-Inflamatórios/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Edema/prevenção & controle , Humanos , Quinase I-kappa B , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular , Piridinas/síntese química , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes, CRTH2, is a cognate receptor for prostaglandin (PG) D(2) and, in humans, is suggested to play a functional role in Th2-dependent allergic inflammation. While peripheral blood leukocytes expressing high levels of surface CRTH2 have been detected in disease, little is known of the functional significance of CRTH2 in disease etiology. We have utilized a Th2-dependent murine model of FITC-induced contact hypersensitivity to assess the role, if any, CRTH2-PGD(2) may play in the elicitation or maintenance of such pathobiology. Expression of both PGD(2) and CRTH2 in lesional skin was paralleled by the release of the chemoattractants LTB(4) and the chemokine KC, as well as a profuse dermal neutrophilic and eosinophilic infiltrate, closely paralleling the acute inflammatory pathology observed in human atopic dermatitis. A small molecule CRTH2 antagonist, but not a selective PGD(2)R (DP) receptor antagonist, was able to completely abrogate these responses. Inflammatory cascades mediated by CRTH2 ligation may therefore represent an important early step in the elicitation and maintenance of Th2-dependent skin inflammation.
Assuntos
Dermatite Alérgica de Contato/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Prostaglandina D2/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Células Th2/imunologia , Animais , Carbazóis/administração & dosagem , Quimiocina CXCL1 , Quimiocinas , Quimiocinas CXC , Citocinas/imunologia , Dermatite Alérgica de Contato/patologia , Dermatite Atópica/imunologia , Eosinófilos/imunologia , Feminino , Inflamação/imunologia , Inflamação/patologia , Leucotrieno B4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ativação de Neutrófilo/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/administração & dosagemRESUMO
Contact hypersensitivity (CHS) induced by a hapten is thought to be mediated by T helper type 1 (Th1) cells. However, FITC can induce contact allergy in vivo, and in vitro studies suggest that this response is Th2-type driven. We compared CHS reactions induced by FITC or dinitrofluorobenzene (DNFB), a well-known Th1 inducing hapten, in Balb/c mice, C57/B6 mice, and several gene knock-out mice, and investigated the role of Th1/Th2 cytokines, T cell populations, eosinophils, and mast cells. Balb/c mice (Th2 dominant strain) had a stronger response to FITC than C57/B6 mice (Th1 dominant strain). The skin inflammation was characterized by edema and eosinophilia, and serum IgE levels were elevated following FITC challenge. All responses were enhanced by a second round of sensitization. Anti-TNF-alpha or anti-very late antigen-4 (VLA-4) antibody partly inhibited both FITC- and DNFB-induced CHS. Pretreatment of mice with anti-IL-4 antibody, anti-IL-5 antibody, recombinant INF-gamma, or the mast-cell depleting agent 48/80 significantly diminished edema formation, and Stat6(-/-) mice were fully protected from FITC-induced CHS, while DNFB-induced CHS was enhanced (Stat6(-/-), mast cell depletion) or not affected (anti-IL-5 antibody). Further, mice lacking CD4(+) T cells and mice lacking both CD8 and MHC II showed very little reaction at all to FITC, while the absence of CD8 T cells alone or MHC II alone conferred partial protection only. These findings indicate a contribution of MHC II-independent CD4(+) T cells and/or CD4(+) NKT cells to the Th2 response triggered by FITC in vivo, and makes FITC-induced CHS a suitable animal model for atopic dermatitis.