RESUMO
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.
Assuntos
Luciferases/metabolismo , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/farmacologia , Rolipram/farmacologia , Linhagem Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Humanos , Luciferases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , TransfecçãoRESUMO
Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.