Screening And Functions Detection Of Small Molecule C5a Antagonists/Agonists

Objective To screen some effective and with new structures antagonists and agonists of C5a, through using the virtual screen and the our new established, stable methodology in whole human blood for screening antagonists and agonists of C5a. Methods Whole blood from volunteer was collected and stimulated with C5a at different concentrations 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 neutrophil,including lysozyme release,oxidative burst and cytokine IL-8 production,were also examined respectively. Finally,western blotting was used to detect the content of total and phosphorylated ERK and AKT. Those detections are to investigate the effects about C5a stimulant and the positive medicine PMX-53 on biochemical indicators. The experiment platform for screening C5a antagonists and agonists was establishment by using some ways such as flow cytometry, lysozyme release, oxidative burst, ELISA, western blotting ,and so on. While on the basis of 3-D crystal structure of C5a and complex model of C5a-substrate analogue, molecular docking-based virtual screening against the catalytic domain of C5a was performed on a SPECS 3D database that ~200,000 commercially available organic compounds had been built via in-house procedures. Top 2000 compounds with the best energy score were selected for further diversity analysis. From different structurally diverse clusters, 131 compounds were selected for biological assay based on physicochemical properties, chemical stability, potential toxicity and potential metabolism, and so on. Those 131 compounds through virtual screening and reserved medicine which we had were screening by using the experiment platform in vitro. Results C5a stimulation actively up-regulated CD11b expression,evoked lysozyme and reactive oxygen species release,increased IL-8 production and levels of total and phosphorylated ERK/AKT. Addition of the positive medicine PMX-53 significantly blocked these effects. It demonstrated that the experiment platform for screening C5a antagonists/agonists was established successfully. While on the basis of 3-D crystal structure of C5a and complex model of C5a-substrate analogue, molecular docking-based virtual screening against the catalytic domain of C5a was performed on a SPECS 3D database that ~200,000 commercially available organic compounds had been built via in-house procedures. At last, 131 compounds were selected.Some small compounds which we have and these 131 compounds through the vitro screening can blocked or cooperation these C5a effects. Conclusion Using the computer virtual screen, 131 compounds were selected. Otherwise we successfully established the vitro methodology for screening C5a antagonists in human whole blood. Using this methodology, we found some small compounds from which we have and others which through the vitro screening can inhibit or up-regulation these C5a-C5aR effects. This will be a basement for screening new and effective C5a antagonists/agonists.