FP Assay for Screening Compounds Targeting RORα and RORγ

We employed FP assay to evaluate the library of compounds in our laboratory. In this assay, we utilized a fluorescence probe that was FITC-labeled 25-OH-cholesterol, which is a natural ligand with a high affinity for RORα (Kd = 3.3 ± 0.89 nM) and RORγ (Kd = 5.1 ± 0.71 nM). When the fluorescence probe interacts with the protein RORα-LBD or RORγ-LBD (Fig. 8), the relative molecular mass increases, the free rotation rate reduces, and a high FP value is generated. If the test compounds can bind to the LBD, they will compete with the fluorescence probe, leading to a lower FP value than without the test compounds due to a smaller relative molecular mass and a faster free-spin rate. In a competitive binding assay using FITC-labeled 25-hydroxycholesterol (Fig. 9), we found that 7,9-octadecadienoic acid ester of CSL, 28-OH-CSL, exhibited the best binding to the RORα-LBD region with affinity Kd values of 0.28 and 0.29 μM. 12-OH-CSL, a-ring aromatized 15-OH-CSL, and the RORγ-LBD region showed the best binding, with Kd values of 0.22 μM.