DC646 antagonizes FXR via a novel molecular mechanism. (a) Strategy of the AlphaScreen assay in (b). (b) IC50 of DC646 in recruiting the peptides of SRC2-3 under different adding orders of DC646, adding DC646 after CDCA, DC646 combined with CDCA, or CDCA after DC646, which have been incubated with His-hFXRα-LBD, to test its ability to inhibit recruitment of the SRC2-3 peptide (n = 3 in each group). (c) DC646 and CDCA bind to FXR-LBD by different binding sites. BIOTIN-DC646 (the synthesis of BIOTIN-DC646 is described in Supporting Information, Supplementary Fig. S4) and the purified FXR-LBD were incubated together with or without CDCA or DC646. These mixtures were immunoprecipitated with His beads and then immunoblotted with anti-BIOTIN antibodies. (d) DC646 antagonizes FXR by interfering FXR-LBD with the coactivator. In the existence of CDCA and the purified FXR-LBD, BIOTIN-DC646 was incubated with or without SRC2-3. These mixtures were immunoprecipitated with His beads and then immunoblotted with anti-BIOTIN antibodies. (e) DC646 antagonizes FXR by binding to the surface of FXR-LBD, thereby inhibiting the binding of FXR-LBD and coactivators. In the existence of SRC2-3 and the purified FXR-LBD, BIOTIN-DC646 was incubated with CDCA in different adding orders indicated by (a). These mixtures were immunoprecipitated with His beads and then immunoblotted with anti-BIOTIN antibodies.
