Molecular Mechanics Generalized Born Surface Area (MMGBSA) and Fluorescence Polarization (FP) Competitive Binding Experiment for RORα and RORγ Ligand Binding

The MMGBSA method was used to calculate the free energy of binding after stabilizing the system. The OPLS3e force field was utilized for the minimization of the receptor and ligand complexes, with the local optimization features in Prime. Ligand strain energies were also calculated during MMGBSA. The complexes were investigated for residues at sites involved in molecular mechanics.

For the FP competitive binding experiment, human RORα-LBD (271-513) and RORγ-LBD (262-507) were constructed onto N-terminal His-tagged pET28a vectors and transformed into E. coli BL21 (DE3). The bacterial precipitates were lysed and the supernatant was collected and purified using a Ni2+ affinity column. For the FP experiments, the solutions used were pH 7.4, 50 mM HEPES, 0.01% bovine serum albumin, 150 mM NaCl, and 5 mM MgCl2 in a corning NBS black 96-well plate with a final concentration of 150 nmol/L RORα or RORγ and 5 nmol/L fluorescein-labelled 25-hydroxycholesterol. The compounds were incubated for 12 h at 37 °C, and the plates were read using a Biotek SynergyH1 Multifunctional Enzyme Labeler. The results were analyzed using GraphPad Prism and the Cheng-Prusoff equation was used to determine the Kd.