DC646 exerts FXR antagonistic effects by interacting with coactivator via Q296 and K303. (a) Strategy of the AlphaScreen assay in (b) and (c). (b and c) Various concentrations of Ivermectin are incubated with His-hFXRα-LBD to obtain the EC₅₀ of Ivermectin in recruiting the peptides of SRC2-3 (b) or NcoR2 (c) (n = 3 in each group). (d and e) DC646 at 11 μmol/L is incubated with His-hFXRα-LBD to test its inhibitory ability in recruiting the peptide SRC2-3 (d) or NcoR2 (e) in the presence of Ivermectin (n = 3 in each group). (f) Molecular docking model of DC646 to FXR-LBD (PDB code: 4WVD). The key residues Q296 and K303 and DC646 are shown as sticks. (g) Effects of FXR key residue mutations on its transcriptional activity in response to DC646 by reporter assay (n = 3 in each group). Data are presented as mean ± SEM and analyzed by one-way ANOVA with Dunnett’s post hoc test for (d) and (e). ^***P ≤ 0.001.