Jie Zhang,Tiezhu Li,Tuoyi Wang,Tianzhu Guan,Hansong Yu,Zhuolin Li,Yongzhi Wang,Yongjun Wang,Tiehua Zhang
The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD) was examined by a combination of in vitro investigation and in silico simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential (ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the positive electrostatic potential and thereby have a significant influence on the electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also contribute to stabilize the binding of the halogenated BPAs to their receptor molecule. Comparison of the calculated binding energies with the experimentally determined affinities yielded a good correlation (R 2 = 0.6659) that could provide a rational basis for designing environmentally benign chemicals with reduced toxicities. This work can potentially be used for preliminary screening of halogenated BPAs.
Jie Zhang,XiaoJia Xing,Yonghai Sun,Zhuolin Li,Peiyu Xue,Tuoyi Wang,Tiezhu Li
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 (Kd = 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.
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