RESUMO
A structural survey of pyrazolopyridine-pyridazinone phosphodiesterase (PDE) inhibitors was made with a view to optimization of their dual PDE3/4-inhibitory activity for respiratory disease applications. These studies identified (-)-6-(7-methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridine-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490, compound 2ac) as a compound with potent combined bronchodilatory and anti-inflammatory activity and an improved therapeutic window over roflumilast.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Broncodilatadores/farmacologia , Desenho de Fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Broncodilatadores/síntese química , Broncodilatadores/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Cobaias , Estrutura Molecular , Inibidores da Fosfodiesterase 3/síntese química , Inibidores da Fosfodiesterase 3/química , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/química , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas/fisiologia , Linhagem Celular , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/química , Genes Reporter , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana , Proteínas Associadas aos Microtúbulos/química , Antígenos de Histocompatibilidade Menor , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fes/química , Proteínas Proto-Oncogênicas c-fes/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Aurora-A is known to be a mitotic kinase required for spindle assembly. We constructed a human stable cell-line in which Aurora-A, histone H3 and importinalpha were differentially expressed as fusions to green, cyan, and red fluorescent proteins (GFP, CFP and DsRed). In interphase cells, GFP-Aurora-A was localized in the centrosome. Its molecular behavior in living mitotic cells was extensively analyzed by an advanced timelapse image analyzing system. In G2 phase, duplicated centrosomal dots of Aurora-A separated and moved to the opposite poles, a process requiring 18 min. In prophase, the Aurora-A dots approached closer and the nuclear membrane of DsRed-importinalpha beneath them became thick and invaginated, resulting in a "dumb-bell" shaped nucleus with condensed chromatin. As the importinalpha membrane further shrank and disappeared, the condensed chromatin was excluded from the nucleus and the Aurora-A dots grew rapidly into a spindle-like structure. Congression of mitotic chromosomes continued for 20-50 min until they were properly aligned at the spindle equator and then the sister chromatids started to segregate, taking 4-6 min for them to reach the poles. An importinalpha membrane reappeared around the surface of chromatin 10 min after anaphase onset. Aurora-A gradually decreased in size in telophase and returned to the surface of the newly formed small sister nuclei. These observations showed that the morphological change of Aurora-A was cooperated with the breakdown and reformation of nuclear membrane. Immunostaining with anti-alpha or gamma-tubulin further indicated that Aurora-A was involved in the formation of mitotic spindle in metaphase as well as the subsequent chromosome movement in anaphase.
