Characterization of the binding between phthalate esters and mouse PPARα for the development of a fluorescence polarization-based competitive binding assay

Abstract

The binding of phthalate esters (PAEs) to peroxisome proliferator-activated receptor α (PPARα) has been investigated by using fluorescence polarization (FP) in combination with molecular modeling techniques. The FP competitive binding assay is based on a mouse-derived recombinant PPARα ligand binding domain (LBD) and a fluorescent-labeled fatty acid (C4-BODIPY-C9). A soluble mPPARα-LBD protein derivative, named mPPARα-LBD*, was expressed and purified. By using C4-BODIPY-C9 as a probe, 10 common PAEs with different carbon chain lengths and functional groups were assessed for their binding affinities with mPPARα-LBD*, respectively. PAEs displace the probe from the C4-BODIPY-C9–mPPARα-LBD* complex, resulting in lower polarization values. FP assay showed that PAEs compete for the C4-BODIPY-C9 binding sites in a concentration-dependent manner, and the potency of the tested PAEs increases with increasing side chain length. Molecular docking suggested that the length and hydrophobicity of the side chain of PAEs have contributed a lot to the ligand–receptor binding, and there are four prominent interactions observed to stabilize the PAEs–mPPARα-LBD* binding. In addition, comparison of docking scores vs. experimental binding affinities yielded a good correlation (R² = 0.948). The most active DEHP (K_d = 19.6 ± 1.7 μM) has the lowest ranking on docking scores. The fluorescence polarization-based competitive binding assay can potentially be used for high-throughput screening of PAEs, which may serve as an assistant of chromatographic techniques.