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Citation
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HERO ID
3350287
Reference Type
Journal Article
Title
Characterization of the binding between phthalate esters and mouse PPAR alpha for the development of a fluorescence polarization-based competitive binding assay
Author(s)
State University of New York :: SUNY
Year
2016
Is Peer Reviewed?
1
Journal
Analytical Methods
ISSN:
1759-9660
EISSN:
1759-9679
Publisher
Royal Society of Chemistry
Volume
8
Issue
4
Page Numbers
880-885
Language
English
DOI
10.1039/c5ay03053f
Web of Science Id
WOS:000368943700023
URL
https://search.proquest.com/docview/2237551080?accountid=171501
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Abstract
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.
Keywords
article; binding capacity; binding sites; chromatography; computer simulation; fatty acids; fluorescence; fluorescent labeling; hydrophobicity; ligands; moieties; molecular models; peroxisome proliferator-activated receptors; phthalates; screening
Tags
IRIS
•
Dibutyl Phthalate (DBP)
Database Searches
Litsearch Jan 2016 - July 2016
WOS
LitSearch Jul 2016 - Jan 2017
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Studies with Supporting Data
Mechanistic and genotoxicity studies
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