RESUMO
The synthesis of two series of 4'-aza-carbocyclic nucleosides are described in which the 4'-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A(2A) receptor have been identified from both series. The propionamides 14-18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4'-reversed amide and 4'-N-bonded heterocyclic series, respectively.
Assuntos
Agonistas do Receptor A2 de Adenosina , Compostos Aza/síntese química , Ácidos Carboxílicos/síntese química , Nucleosídeos/síntese química , Nucleotídeos de Pirimidina/síntese química , Animais , Compostos Aza/metabolismo , Compostos Aza/farmacologia , Células CHO , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/farmacologia , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , Nucleotídeos de Pirimidina/metabolismo , Nucleotídeos de Pirimidina/farmacologia , Ratos , Receptor A2A de Adenosina/metabolismoRESUMO
Oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. Histone deacetylase-2 (HDAC2), a critical component of the corticosteroid anti-inflammatory action, is impaired in lungs of patients with COPD and correlates with disease severity. We demonstrate here that curcumin (diferuloylmethane), a dietary polyphenol, at nanomolar concentrations specifically restores cigarette smoke extract (CSE)- or oxidative stress-impaired HDAC2 activity and corticosteroid efficacy in vitro with an EC(50) of approximately 30 nM and 200 nM, respectively. CSE caused a reduction in HDAC2 protein expression that was restored by curcumin. This decrease in HDAC2 protein expression was reversed by curcumin even in the presence of cycloheximide, a protein synthesis inhibitor. The proteasomal inhibitor, MG132, also blocked CSE-induced HDAC2 degradation, increasing the levels of ubiquitinated HDAC2. Biochemical and gene chip analysis indicated that curcumin at concentrations up to 1 muM propagates its effect via antioxidant-independent mechanisms associated with the phosphorylation-ubiquitin-proteasome pathway. Thus curcumin acts at a post-translational level by maintaining both HDAC2 activity and expression, thereby reversing steroid insensitivity induced by either CSE or oxidative stress in monocytes. Curcumin may therefore have potential to reverse steroid resistance, which is common in patients with COPD and asthma.
Assuntos
Corticosteroides/fisiologia , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Curcumina/farmacologia , Histona Desacetilases/metabolismo , Monócitos/efeitos dos fármacos , Oxidantes/farmacologia , Proteínas Repressoras/metabolismo , Fumaça/efeitos adversos , Corticosteroides/farmacologia , Cicloeximida/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Histona Desacetilase 2 , Inibidores de Histona Desacetilases , Humanos , Monócitos/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Inibidores da Síntese de Proteínas/farmacologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Proteínas Repressoras/antagonistas & inibidores , Fumar/efeitos adversos , Nicotiana , Células U937RESUMO
A series of novel corticosteroid derivatives featuring C-17 furoate ester functionality have been synthesised. Profiling in vitro and in vivo has resulted in the identification of a compound with a longer duration of action and a lower oral side effect profile in rodents compared to budesonide.
Assuntos
Androstenos/síntese química , Ésteres/síntese química , Glucocorticoides/síntese química , Receptores de Glucocorticoides/agonistas , Androstenos/farmacocinética , Androstenos/farmacologia , Animais , Disponibilidade Biológica , Linhagem Celular , Eosinofilia/tratamento farmacológico , Ésteres/farmacocinética , Ésteres/farmacologia , Glucocorticoides/farmacocinética , Glucocorticoides/farmacologia , Humanos , Macrófagos/citologia , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Timo/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.
Assuntos
Proteínas da Matriz Extracelular/fisiologia , Matriz Extracelular/fisiologia , Macrófagos/fisiologia , Neutrófilos/citologia , Fumar/efeitos adversos , Aldeídos/farmacologia , Apoptose/fisiologia , Adesão Celular/efeitos dos fármacos , Colágeno Tipo IV/fisiologia , Fibronectinas/fisiologia , Humanos , Receptores de Hialuronatos/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Macrófagos/metabolismo , Neutrófilos/metabolismo , Fagocitose , Fumar/patologia , Células U937RESUMO
Pulmonary emphysema in chronic obstructive pulmonary disease (COPD) is characterized by the destruction of the alveolar walls leading to permanent enlargement of distal respiratory air spaces. A major causal factor is cigarette smoking, which produces conditions of chronic oxidative stress within the lungs. At a cellular level, increased macrophage accumulation and retention within the alveolar interstitial spaces is pivotal to the development of emphysema. To date it has been unclear as to the underlying mechanisms relating chronic oxidative stress to macrophage accumulation and retention. Our study was initiated to ascertain the role of modification of extracellular matrix proteins with cigarette smoke and products of lipid peroxidation on macrophage adhesion and activation. Increased numbers of macrophages were seen adhering to cigarette smoke-modified collagen IV as compared to unmodified collagen, where little or no adherent macrophages were observed. Similar observations were made when collagen was modified with either acrolein or 4-hydroxy-2-nonenal. Adhesion could be blocked with either fucoidan or a monoclonal antibody against the Type A macrophage scavenger receptor. Also, modified collagen triggered both oxidative burst and MCP-1 release in macrophages. These results, therefore, highlight a potential mechanism by which oxidative stress through the production of reactive carbonyls promotes macrophage accumulation, retention, and activation, independently of other proinflammatory stimuli. The implications of this for the development of emphysema in COPD are discussed.
Assuntos
Peroxidação de Lipídeos , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Fumar/efeitos adversos , Acroleína/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Estresse Oxidativo , Plantas Tóxicas , Proteínas/química , Proteínas/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Enfisema Pulmonar/etiologia , Receptores Imunológicos/biossíntese , Receptores Depuradores , Fumaça/efeitos adversos , NicotianaRESUMO
We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.