Discovery of cyclopeptide as FXR antagonist. (a) Structures of FXR-LBD complex with agonists (CDCA and GW4064), an antagonist (Ivermectin), and coactivator. The crystal structures (PDB codes: 3DCT, 4WVD, and 6HL1) were structurally aligned with respect to LBD. Agonists and antagonist are shown as sticks. FXR and coactivator are displayed in cartoon. (b) Interfaces of FXR for coactivator and AF2. FXR-LBD is shown as surface. (c) The cyclopeptide library has been screened for antagonistic activity of FXR. (d) Illustration of an alpha protein-protein interaction assay (AlphaScreen assay). (e) Strategy of AlphaScreen assay in (f). (f) GUDCA at 50 μmol/L, GlyMCA at 50 μmol/L, and DC644, SJN10, SJN18, HT14, HT15, and HT18-2 at 10 μmol/L are incubated with His-hFXRα-LBD to test its ability to inhibit recruitment of the SRC2-3 peptide in the presence of CDCA (n = 3 in each group). (g) Molecular docking model of DC644 to FXR-LBD (PDB code: 4WVD). Data are presented as mean ± SEM and analyzed by one-way ANOVA with Dunnett’s post hoc test for (f). ^*P ≤ 0.05, ^**P ≤ 0.01, and ^***P ≤ 0.001.