Study On The Drug Target Of Geniposide Based On Bioinformatics And Affinity Chromatography

ObjectiveThe aim of this study is utilizing affinity chromatography technology and bioinformatics methods to screen the specific binding protein of geniposide and laying the foundation for investigate the interactions between geniposide and its target proteins. In addition, it also provides technological means for the other Chinese medicine small molecules analogue with active hydroxyl to screen the drug targets.MethodsThis study include three aspects as follows:First, The ChemBio3D Ultra software was used to plot the structure of geniposide ligand. Using geniposide as a small molecular probe to search potential target proteins from ChemMapper, PharmMapper and TarFisDock drug target database. We adopted a hybrid similarity metric algorithm combined with molecular shape and three-dimension pharmacophore features, named as SHAFTs, to calculate and rank the docking results based on lamarckian genetic algorithms and free-energy scoring function. Second, ECH, the abbreviation for Epichlorohydrin, was adopted as activating reagent for surface modification of agarose gel Sepharose CL-6B. Geniposide was coupled with epoxy activated Sepharose CL-6B by hydroxyl-epoxy interaction, and high performance liquid chromatography was put into use to verify the efficiency of immobilization. Then geniposide-epoxy activated Sepharose CL-6B was applied to screening the binding proteins of geniposide in mouse brain tissue and peripheral blood mononuclear cell. Third, peripheral blood mononuclear cell was incubated with geniposide and fluorescein isothiocyanate (FITC) labelled dexamethasone in order to detect the drug uptake via flow cytometry and preliminarily validate whether there is the interaction between geniposide and glucocorticoid receptor.Results1. Virtual screening from drug-target database predicted that glucocorticoid receptor may one of the binding protein of geniposide, and the results of molecular docking showed that the Total Score between geniposide and glucocorticoid receptor were 10.56, Crash was-1.27, and polar is 3.59.2. The optimal condition of epoxy activated Sepharose CL-6B were:15% ECH,0.8 mol/L NaOH at 37℃ for 3 h. After optimized the conditions, the epoxy group density was up to 122 μmol/mL3. Geniposide was immobilized with epoxy activated Sepharose CL-6B by hydroxyl-epoxy interaction, the conjugation ratio and amount with high performance liquid chromatography were 19.53% and 17.08 μmol/mL, respectively.4. By affinity chromatography, multiple protein bands were identified in SDS-PAGE, and then mass spectrometry results indicated that bands were comprised by glucocorticoid receptor, heat shock protein 70, heat shock protein 90 and brain acid soluble protein.5. Flow cytometry showed that the mean fluorescence intense of dexamethasone significantly decreased in peripheral blood mononuclear cell after addition of the geniposide and in dose dependent manner.Conclusions1. Through ligand-based model of virtual screening and molecular docking, we predicted that glucocorticoid receptor may be the binding proteins of geniposide. Geniposide could match into glucocorticoid receptor and the binding pocket may locate in the intramolecular.2. We established the optimal condition of epoxy activated Sepharose CL-6B, and successfully prepared the geniposide-epoxy activated affinity resin that was applied to screening the binding proteins of geniposide from mouse brain tissue and peripheral blood mononuclear cell.3. Drug uptake assay manifested that geniposide could competitive combined with glucocorticoid receptor with dexamethasone, which suggested that there exists certain interaction between glucocorticoid and glucocorticoid receptor.