US EPA

3350287 

Journal Article 

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

State University of New York :: SUNY 

2016 

1 

Analytical Methods
ISSN: 1759-9660
EISSN: 1759-9679 

Royal Society of Chemistry 

8 

4 

880-885 

English 

10.1039/c5ay03053f 

WOS:000368943700023 

https://search.proquest.com/docview/2237551080?accountid=171501
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The binding of phthalate esters (PAEs) to peroxisome proliferator-activated receptor alpha (PPAR alpha) 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 PPARa ligand binding domain (LBD) and a fluorescent-labeled fatty acid (C4-BODIPY-C9). A soluble mPPAR alpha-LBD protein derivative, named mPPAR alpha-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 mPPAR alpha-LBD*, respectively. PAEs displace the probe from the C4-BODIPY-C9-mPPAR alpha-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-mPPAR alpha-LBD* binding. In addition, comparison of docking scores vs. experimental binding affinities yielded a good correlation (R-2 = 0.948). The most active DEHP (K-d = 19.6 +/- 1.7 mu M) 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. 

article; binding capacity; binding sites; chromatography; computer simulation; fatty acids; fluorescence; fluorescent labeling; hydrophobicity; ligands; moieties; molecular models; peroxisome proliferator-activated receptors; phthalates; screening