RESUMEN
In this study, we used the tumor immunotherapy protein indoleamine 2,3-dioxygenase 1 (IDO1) as the target, and proposed an enzyme-cell-based tertiary IDO1 inhibitor high throughput screening platform. Firstly, the recombinant human IDO1 protein was expressed by genetic engineering and efficient IDO enzymatic screening system was established. Secondly, A172 cells stimulated with interferon-γ (IFNγ) or constructed plasmid which could highly express human IDO1 protein in HEK293 cells with transient transfection were used to construct the specific IDO1 cell based screening system. Finally, the effect of the compound on kynurenine and tryptophan in mouse plasma was determined by LC/MS/MS method on C57 mice, which could further verify the inhibitory effect of the selected compounds on IDO1 in vivo. The established and optimized enzyme-cell based screening model in this study can efficiently and effectively obtain IDO1 inhibitors in vitro, which lays a good foundation for the rapid development of clinical drugs. Procedures for animal study were performed with approval of the Animal Care and Use Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.
RESUMEN
Objective: To establish the stable methodology for screening antagonists of CD88, one of C5a receptors. Methods: Whole blood from volunteers was collected and then stimulated by C5a with different concentration for 10-30 min in the presence or absence of PMX-53 and LPS. Flow cytometry was used to detect the expression of CD11b which on the surface of neutrophil. Other functions of nentrophil, including lysozyme release, oxidative burst and interleukin (IL)-8 production, were also examined respectively. Finally, western blotting was used to detect the content of total and phosphorylated ERK and AKT. Thus, the effects about C5a receptor stimulant and the positive medicine PMX-53 on biochemical indicators were investigated. Results: The C5a stimulation actively up-regulated CD11b expression, evoked lysozyme and reactive oxygen species release, increased the IL-8 production and the contents of total and phosphorylated ERK/AKT. While the positive medicine PMX-53 significantly blocked these effects. Conclusion: The methodology in vitro for screening C5a receptor antagonists inhuman whole blood is successfully established.