The Establishment And Application Of A High-throughput Drug Screening Model For FXR Agonists

FXR agonists have been demonstrated a critical role for FXR in maintaining cholesterol and bile acid homeostasis, and FXR has become a valuable target for the identification of novel drugs to treat dyslipidaemia and cholestasis. Regulation of FXR through small-molecule drugs represents a promising therapy for diseases resulting from lipid, cholesterol and bile acid abnormalities.To find the new lead compounds for dyslipidaemia or cholestasis drug from the microbial secondary metabolites library, a high-throughput screening model for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system was developed.The cDNA encoding the human FXR ligand binding domain (DBD) was amplified by RT-PCR from liver cell total mRNA and fused to the DNA binding domain of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid.Five copies of the GAL4 DNA binding site was inserted into upstream of the SV40 promoter of pGL3-promoter Vector to construct a reporter plasmid PGL3-GAL4.A stable and sensitive cell-based high-throughput screening model for FXR agonists was established. After optimization, CDCA, a FXR natural ligand, induced the expression of luciferase gene in a dose-dependent manner with high signal/noise ratio. And 120 compounds and 880 samples of microbial secondary metabolites library were screened using this HTS model. CoQ10 and F01WA2076A were screened out and proved to be two agonists of FXR.In addition, 8896 samples of secondary metabolites from Fungi and Actinomyces were prepared for high-throughput screening models.The HTS model for FXR agonists and two agonists (CoQ10 and F01WA2076A) will contrubute to the development of novel drugs and the study for FXR physiological mechanism.