Fig. 1.
INT-767 is a potent FXR agonist. A, ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay. hFXR-LBD-GST was incubated with increasing concentrations of the indicated ligands in the presence of biotinylated Src-1 peptide. The AlphaScreen signal increases when the complex receptor-coactivator is formed. EC50 values were 7 μM, 76 nM, and 7 nM for CDCA, INT-747, and INT-767, respectively. The results show mean ± S.D. of triplicate samples from a representative experiment of 10 performed. B, transactivation assay on HepG2 cells performed by transient transfection of full-length FXR and the canonical FXRE containing 3 inverted repeats (IR1). Vector only, empty vector-transfected cells treated with 10 μM concentrations of the indicated compounds. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. C, transactivation assay in HEK293T cells by using Gal4-FXR-LBD and (UAS)5-Luc system. Vector only: empty vector-transfected cells treated with 10 μM INT-767. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. D to G, regulation of FXR target genes assessed by quantitative RT-PCR. INT-767 increases Bsep, Ost, and Shp and downregulates Cyp7α1 mRNA expression. The results show mean ± S.D. of triplicate samples from a representative experiment of three performed. *, p < 0.05; **, p < 0.001 versus vector only or control, cells stimulated with medium only.