4.2. AlphaScreen Co-Regulators Binding Assay

The binding of multiple co-regulator motif peptides to FXR-LBD protein in response to compounds was determined by AlphaScreen assay (the schematic diagram is shown in Figure 1B) using a hexahistidine detection kit (Perkin-Elmer, code 6760619R). For screening (natural product library from BioBioPha company, China. http://www.biobiopha.com/ (accessed on 14 November December 2022)), small molecules were transferred into a 384-well OptiPlate by Echo520 (Beckman Coulter, Inc. California, USA), the mixture containing FXR-LBD and biotin-SRC2-3 was transferred into the OptiPlate wells by a multidrop microplate dispenser (Thermo Fisher, USA), and AlphaScreen signaling was measured using an Envision reader (Perkin-Elmer, USA) after incubation for 1 h at room temperature. The assay was performed using approximately 20~40 nM 6His-FXR-LBD protein and approximately 20 nM biotinylated co-regulator motif peptides in the presence of 2~5 μg/mL donor beads (streptavidin-coated) and acceptor beads (nickel-coated) in a buffer containing 50 mM MOPS, 50 mM NaF, 0.05 mM CHAPS, and approximately 0.1 mg/mL BSA, pH 7.4. The following biotinylated co-regulator motif peptides were used:

SRC1-2, biotin-SPSSHSSLTERHKILHRLLQEGSP

SRC2-3, biotin-QEPVSPKKKENALLRYLLDKDDTKD

NCoR-2, biotin-GHSFADPASNLGLEDIIRKALMGSF