FP Assay Screening of Compound Library for RORα and RORγ Ligands

The library of compounds in our laboratory was screened using the FP assay. This involved using a fluorescence probe for FITC-labeled 25-OH-cholesterol, which has high affinity for RORα (Kd = 3.3 ± 0.89 nM) and RORγ (Kd = 5.1 ± 0.71 nM) as a natural ligand. When the fluorescence probe interacts with the protein RORα-LBD or RORγ-LBD (as shown in Fig. 8), the relative molecular mass becomes larger, the free rotation rate slows down and a high FP value is generated. If the test compounds can bind to the LBD, they will compete with the fluorescence probe, resulting in a smaller relative molecular mass and a faster free-spin rate, producing a lower FP value than without the test compounds. In a competitive binding assay with FITC-labelled 25-hydroxycholesterol (as shown in Fig. 9), 7,9-octadecadienoic acid ester of CSL, 28-OH-CSL had the best binding affinity to the RORα-LBD region with Kd values of 0.28 and 0.29 μM. 12-OH-CSL, a-ring aromatized 15-OH-CSL and the RORγ-LBD region had the best binding, both with Kd values of 0.22 μM.