RESUMO
The synthesis and biological activities of imidazo[5,1-f][1,2,4]triazin-2-amines (imidazotriazines) as novel polo-like kinase 1 inhibitors are reported.
Assuntos
Aminas/síntese química , Aminas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Imidazóis/síntese química , Imidazóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Triazinas/síntese química , Administração Oral , Aminas/química , Animais , Antineoplásicos/química , Técnicas de Química Combinatória , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Imidazóis/química , Camundongos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Triazinas/química , Triazinas/farmacologia , Quinase 1 Polo-LikeRESUMO
Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine- and stress-induced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The Km of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.