Binding interactions of halogenated bisphenol A with mouse PPARα: In vitro investigation and molecular dynamics simulation

Accepted Manuscript Title: Binding interactions of halogenated bisphenol A with mouse PPAR␣: in vitro investigation and molecular dynamics simulation Authors: Jie Zhang, Tiezhu Li, Tuoyi Wang, Tianzhu Guan, Hansong Yu, Zhuolin Li, Yongzhi Wang, Yongjun Wang, Tiehua Zhang PII: DOI: Reference:

S0378-4274(17)31452-2 https://doi.org/10.1016/j.toxlet.2017.11.004 TOXLET 9995

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

6-9-2017 5-10-2017 5-11-2017

Please cite this article as: Zhang, Jie, Li, Tiezhu, Wang, Tuoyi, Guan, Tianzhu, Yu, Hansong, Li, Zhuolin, Wang, Yongzhi, Wang, Yongjun, Zhang, Tiehua, Binding interactions of halogenated bisphenol A with mouse PPAR␣: in vitro investigation and molecular dynamics simulation.Toxicology Letters https://doi.org/10.1016/j.toxlet.2017.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Binding interactions of halogenated bisphenol A with mouse PPARα: in vitro investigation and molecular dynamics simulation Jie Zhanga,1, Tiezhu Lia,1, Tuoyi Wang a,1, Tianzhu Guanb, Hansong Yuc, Zhuolin Lia,

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Yongzhi Wanga, Yongjun Wanga,*, Tiehua Zhangb,* Institute of Agricultural Resources and Environment, Jilin Academy of Agricultural Sciences,

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Changchun 130033, China

College of Food Science and Engineering, Jilin University, Changchun 130062, China

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College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118,

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China

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* Corresponding authors.

E-mail addresses: [email protected] (Y. Wang), [email protected] (T. Zhang). These authors contributed equally to this work.

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GRAPHICAL ABSTARCT

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+ mPPARα

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Halogenated derivatives of bisphenol A

Fluorescence polarization

Electrostatic potential

Molecular docking

Highlights • Interaction of halogenated BPAs with mPPARα-LBD* was investigated. • Halogenated BPAs are affinity ligands for mPPARα-LBD*. • The electronic properties varied with the halogenation patterns. • The binding modes were illustrated by molecular docking.

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• This work can be used for preliminary screening of halogenated BPAs.

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ABSTRACT

The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs)

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to mouse peroxisome proliferator-activated receptor α ligand binding domain

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(mPPARα-LBD) was examined by a combination of in vitro investigation and in silico

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simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could

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bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential

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(ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the

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positive electrostatic potential and thereby have a significant influence on the

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electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also

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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 (R2 = 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.

Keywords: Fluorescence polarization Electrostatic potential

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Molecular docking Halogenated bisphenol A

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Peroxisome proliferator-activated receptor α

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1. Introduction

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In recent years, great concerns have been raised about human exposure to

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environmental endocrine-disrupting compounds (EDCs). Bisphenol A (BPA), an estrogenic EDC mainly used in the manufacturing of polycarbonate plastics and

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epoxy resins, is among the highest volume chemicals produced around the world

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(Jiménez-Díaz et al., 2010; Vom Saal et al., 2012). Halogenated derivatives of

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bisphenol A (halogenated BPAs), which feature bromine or chlorine substituents on the phenolic rings, are widely used as flame retardants (FRs) to reduce the flammability of polymers (Delfosse et al., 2015). Tetrabromobisphenol A (TBBPA)

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and tetrachlorobisphenol A (TCBPA) are basically the most consumed organic FRs (Chu et al., 2005). Higher halogenated BPAs may be dehalogenated to lower congeners under anaerobic conditions. Consequently, the presence of TBBPA and TCBPA, as well as that of lower halogenated analogues (mono-, di- and

tri-halogenated BPAs) in the environmental samples has been unequivocally demonstrated (Arbeli and Ronen, 2003; Huang et al., 2013). Previous studies have revealed the potential ecological risk of BPA on plants growth and development by inhibiting photosynthesis (Jiao et al., 2017; Sun et al.,

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2013). BPA and its halogenated derivatives can also cause adverse effects on human

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health due to their endocrine disrupting properties (Kitamura et al., 2005; Rubin,

2011). These compounds share some physicochemical properties with natural ligands, allowing them to target the nuclear receptors, such as the estrogen receptors (ERα, β)

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(Matthews et al., 2001), the estrogen-related receptor γ (ERRγ) (Takayanagi et al.,

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2006), the androgen receptor (AR) (Paris et al., 2002), the glucocorticoid receptor

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(GR) (Zhang et al., 2017c), the pregnane X receptor (PXR) (Sui et al., 2012), the thyroid hormone receptors (TRα, β) (Moriyama et al., 2002), and the peroxisome

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proliferator-activated receptor γ (PPARγ) (Riu et al., 2011a; Riu et al., 2011b).

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Therefore, the total impact of bisphenols may be induced by the synergistic actions via various metabolism pathways.

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Three isotypes of PPARs (α, β, and γ) exhibit distinct tissue distributions,

physiological roles and ligand specificity (Zoete et al., 2007). PPARs activate gene

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transcription in response to their natural ligands, which comprise a variety of unsaturated fatty acids such as the essential fatty acids linoleic, linolenic and arachidonic acids (Krey et al., 1997). With a modular structure similar to other members of the nuclear hormone receptor superfamily, PPARγ contains three major

functional domains, namely an N-terminal activation function-1 domain (AF-1), a central DNA-binding domain (DBD) followed by a hinge domain, and a C-terminal ligand-binding domain (LBD) (Kilroy et al., 2009). Based on the crystal structure of human PPARγ, the Y-shaped ligand-binding pocket (LBP) is buried within the bottom

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half of the LBD with a volume of approximately 1440 Å3, which is markedly larger than those observed in other nuclear receptors (Itoh et al., 2008). The crystal

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structures of PPARγ in complex with TBBPA and TCBPA reveal that the compounds basically occupy the β-sheet sub-pocket, with one of the phenol rings nestled between

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H3 and the β-sheet, and only a small part of the AF-2 sub-pocket with no direct

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interaction with H12 (Delfosse et al., 2015). Both TBBPA and TCBPA have been

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identified to be the ligands of human, zebrafish, and Xenopus PPARγ, indicating that these compounds can disrupt the activity of their corresponding receptors from

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different species (Riu et al., 2011a).

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In this regard, most previous studies have focused on the interactions between halogenated BPAs and PPARγ (Riu et al., 2011a; Riu et al., 2011b). Given that the

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expression of the soluble protein of PPARα is not as efficient as that of PPARβ and PPARγ (Devchand et al., 1999), little information is available on the binding of

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bisphenols with PPARα. In our early work, the ligand binding domain of mouse-derived PPARα was expressed predominantly as insoluble inclusion bodies. To improve the efficiency of soluble expression, the receptor was modified with computer-aided molecular modeling design by our lab. The deletion of N-terminal

amino acids from 202 to 266 was conducted to produce a new soluble protein named mPPARα-LBD*. In view of the fact that the recombinant protein derivative contains the critical structure of the ligand binding domain, it is expected that the ligand

been demonstrated by our lab (Zhang et al., 2017a; Zhang et al., 2016).

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binding properties of mPPARα-LBD* is identical to that of mPPARα-LBD, which has

In this work, a combination of in vitro assay and in silico modeling has been

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performed to study the binding of BPA and its halogenated analogues with PPARα. The soluble protein mPPARα-LBD* was prepared and applied in the fluorescence

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polarization assay. It is based on the coupling of receptor and tracer, capable of

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modulating the fluorescence polarization emission on the basis of the binding of

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fluorescent probe to mPPARα-LBD*. In order to explore the effect of halogens such as Br, Cl, and F on the charge distribution of bisphenols, the molecular electrostatic

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potential (ESP) was calculated. Then, molecular docking was performed to examine

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the binding modes between the halogenated BPAs and mPPARα-LBD* at the atomic

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level. Furthermore, a correlation analysis was conducted between the calculated binding energies and the experimentally determined data. 2. Materials and methods

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2.1. Materials

A soluble derivative of mPPARα-LBD, named mPPARα-LBD*, was prepared in

our lab (Zhang et al., 2017a; Zhang et al., 2016). Briefly, the deletion of N-terminal amino acids from 202 to 266 was conducted and then the recombinant protein was

expressed in Escherichia coli strain Rosetta (DE3). Dexamethasone fluorescein (Dex-fl) was purchased from Invitrogen Molecular Probes (Eugene, OR, USA). T4 DNA Ligase, 2×Taq PCR Master Mix, and 100 bp DNA Ladder Marker (Gene Answer) were purchased from Codonx Biotech. Co., Ltd. (Beijing, China).

(BPC),

1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane

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4,4′-isopropylidenediphenol (BPA), 1,1-dichloro-2,2-bis(4-hydroxyphenyl)ethylene (HPTE),

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4,4′-(hexafluoroisopropylidene)diphenol (BPAF), 3,3′,5,5′-tetrabromobisphenol A (TBBPA), and 3,3′,5,5′-tetrachlorobisphenol A (TCBPA) were purchased from

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Sigma-Aldrich (St. Louis, MO, USA), TCI (Tokyo, Japan), and Aladdin (Shanghai,

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China). 3-monobromobisphenol A (monoBBPA), 3,3′-dibromobisphenol A (diBBPA),

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3,3′,5-tribromobisphenol A (triBBPA), 3-monochlorobisphenol A (monoCBPA), 3,3′-dichlorobisphenol A (diCBPA), and 3,3′,5-trichlorobisphenol A (triCBPA) were

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synthesized and purified as described previously (Fukazawa et al., 2001; Zalko et al.,

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2006). The structures of the halogenated BPA analogues (halogenated BPAs) above

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are shown in Fig. 1. All other reagents used were of analytical grade. 2.2. Fluorescence Polarization Assay A fluorescein-labeled dexamethasone derivative (Dex-fl) was employed as tracer to

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evaluate the binding affinities of halogenated BPAs with mPPARα-LBD*. Fluorescence polarization assay was carried out by a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Kyoto, Japan) with excitation at 484 nm and emission at 520 nm through a pair of polarizers. In the direct binding experiments, receptor proteins

with a range of concentrations were titrated versus a fixed concentration of probe (8 nM). The increase of fluorescence polarization values upon the formation of Dex-fl-mPPARα-LBD* complexes was monitored. The dissociation constant of Dex-fl with mPPARα-LBD* was calculated by nonlinear curve fitting (Zhang et al.,

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2017b). Then, in the competitive binding experiments, the probe (8 nM) and receptor

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protein (8 nM) were mixed and then various concentrations of halogenated BPAs were added. Each sample was subjected to the fluorescence polarization assay after being

incubated for 40 min at room temperature. The decrease of fluorescence polarization

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values upon addition of halogenated BPAs was monitored and plotted as a function of

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the concentration of competing ligands. The IC50 values (concentrations of

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compounds that inhibited the binding of receptor by 50%) and dissociation constants (Kd) of halogenated BPAs with mPPARα-LBD* were calculated by nonlinear curve

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fitting (Zhang et al., 2017b). Data analysis was performed using GraphPad Prism 5

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(GraphPad Software, USA).

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2.3. Computation of Electronic Properties The initial structures of bisphenols, consisting of two phenolic rings joined by a

bridging alkyl moiety, were constructed and optimized by GaussView 5.0 and

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Gaussian 09W, respectively. To characterize the influences of halogen atoms on the charge distributions, the electrostatic potential of halogenated BPAs ranging from −0.02 (red) to 0.02 (blue) au was calculated by density functional theory (DFT) at the B3LYP/6-31G(d) level. The colour-scaled electrostatic potential maps were generated

with GaussView 5.0. 2.4. Homology Modeling and Molecular Docking The molecular length and Connolly solvent-excluded volume (CSEV) of halogenated BPAs were calculated using AutoDockTools-1.5.6 and Chem3D Ultra 8.0,

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respectively. Homology modeling and molecular dynamics simulation were

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performed to predict and refine the mPPARα-LBD* model. Automated docking calculations were carried out by AutoDockTools-1.5.6 to explore the binding modes of mPPARα-LBD* with these compounds. The size and the center of the grid box

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were set and the predicted binding energies (kcal mol-1) were calculated based on the

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scoring function of AutoDockTools-1.5.6. For each compound, 10 independent

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docking runs were performed and the one with the lowest binding energy was chosen for analysis. The PyMOL program was used to analyze the intermolecular interactions

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of mPPARα-LBD* with halogenated BPAs.

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3. Results and discussion

3.1. Assessment of Halogenated BPAs Binding Potencies with mPPARα-LBD*

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At the beginning of the assay, Dex-fl and mPPARα-LBD* form a complex, which

rotates slowly and produces a high polarization value. With the introduction of

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halogenated BPAs to the reaction system, the added compounds and the probe compete for the binding sites of mPPARα-LBD*. The displaced Dex-fl, with decreasing molecular volume, rotates quickly and results in a low polarization value. Therefore, the receptor-ligand binding can be investigated by monitoring the

fluorescence polarization signal. The dissociation constant of Dex-fl with mPPARα-LBD* obtained from saturation binding curve was 7.04 ± 0.95 nM (Zhang et al., 2017b). Then, the binding capacity of halogenated BPAs with mPPARα-LBD* was determined quantitatively by

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competitive binding assay. The competitive binding curves were shown in Fig. 1 and

the corresponding IC50 values and Kd values were included in Table 1. All the tested

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halogenated BPAs exhibited dose-dependent binding to mPPARα-LBD* as the

affinity ligands, resulting in the activation of their receptor which would in turn

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adversely affect physiological processes. It has been reported that BPA and its

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derivatives promote ovarian cancer progression by directly inducing cell proliferation

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(Hoffmann et al., 2017). They can also cause pathological changes in many organs, particularly the liver and thyroid (Wojtowicz et al., 2014). TBBPA, with the maximum

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number of Br substituents on the phenolic rings, exhibited the strongest binding to

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mPPARα-LBD*. However, with the maximum number of F substituents on the

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bridging alkyl moiety, BPAF showed the weakest receptor binding. This disparity might be due to the different halogenation sites of these two compounds.

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3.2. Halogenation Pattern Dependence of Electronic Properties To explore the effect of halogenation pattern on the binding between halogenated

BPAs and mPPARα-LBD*, the electrostatic potential of halogenated BPAs in the range of -0.02 to 0.02 au was calculated. As shown in Fig. 2, the electronic properties of the halogenated BPAs varied with the halogenation sites and the number of

halogens. Halogens are reported to have an electron withdrawing effect because of their electronegativity (Mayerhöffer et al., 2012). The most positive electrostatic potential of halogenated BPAs is located on the phenolic hydroxyl group, which is also responsible for the formation of hydrogen bond with mPPARα-LBD* (Fig. 3 and

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Fig. S1). In the case of halogen substituents on the phenolic rings, the red regions

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represent negative ESP, which decreases with the successive substitution of halogens at 3,3’,5,5’-sites as the color of the rings gradually fades off. On the other hand, the color of bridging alkyl moieties gradually changes to blue, indicating the increased

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positive ESP. The halogenation on the bridging alkyl moiety for BPC, HPTE, and

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BPAF leads to different ESP maps as discussed above, because the halogen

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substituents can attract electrons to induce a shift of the electron cloud away from the phenolic rings. The gradually changed color to red on the bridging alkyl moieties

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indicates the increased negative ESP. This is consistent with the fact that the negative

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electrostatic potential increases with the increase of halogen atoms.

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As a result, the observed ESP changes may have an effect on the electrostatic interactions of the halogenated BPAs with the surrounding residues of mPPARα-LBD* to influence the binding capacity of the halogenated BPAs to their

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receptor. The calculated binding energies of the halogenated BPAs with the substituents on the phenolic rings are slightly higher than that of their congeners with the substituents on the bridging alkyl moiety (Table 2). It is suggested that the influence of electron cloud caused by halogens on the phenolic rings is stronger than

that on the bridging alkyl moiety (Zhuang et al., 2014), which might be helpful in explaining the calculated energy disparity between two halogenation patterns. 3.3. Molecular Docking of Halogenated BPAs with mPPARα-LBD* The interactions between ligands and receptors are the key factors to govern the

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receptor activities. Herein, an in silico approach was employed to investigate the

molecular recognition between a series of halogenated BPA analogues and

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mPPARα-LBD*. Previous studies have revealed that the ligand-binding pocket of

PPARs forms a Y-shaped cavity with the volume of 1300 to 1400 Å3. The cavity

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consists of a stalk extending from protein surface then branching off to the right

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(channel I) and left (channel II) arms, each of which is ~12 Å in length (Zoete et al.,

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2007). As two representative compounds with halogen substituents on the bridging alkyl moiety and the phenolic rings, Fig. 3 displays the conformations of BPAF and

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TBBPA in the hydrophobic pocket of mPPARα-LBD*, which is obviously large

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enough to accommodate the halogenated BPAs (Table 2). Furthermore, the molecular

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length of other compounds is also less than that of the cavity and thereby they can fit into the channels in a stretched state. The halogenated BPAs adopt a U-shaped conformation that allows the carboxylate group to form hydrogen bonds with the key

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amino acid residues. Due to the bulky halogen substituents, the binding mode of TBBPA is highly similar to that of TCBPA (Fig. 3 and Fig. S1). However, the lower halogenated analogues (mono-, di- and tri-halogenated BPAs) exhibits a different binding mode as compared to the higher congeners (Fig. S1).

Molecular docking analysis revealed that hydrophobic, electrostatic, and hydrogen-bonding interactions were the dominant forces to stabilize the binding of the halogenated BPAs with mPPARα-LBD*, which implied the PPAR activities of these compounds. Several amino acid residues in hydrophobic channels involve in the

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formation of the hydrophobic interactions with the halogenated BPAs, which help to

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improve the stability of receptor-ligand binding. In addition, the halogenated BPAs

contain halogen atoms that also contribute to ligand binding through electrostatic interactions as discussed above. For the halogenated BPAs with the substituents on the

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phenolic rings, the lower halogenated congeners are engaged in two hydrogen bonds

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with mPPARα-LBD*, while only one hydrogen bond is observed between higher

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halogenated congeners (TBBPA and TCBPA) and the receptor. As shown in Fig. S1 and Table 2, most of the halogenated BPAs form hydrogen bonds with Ser89 and

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Tyr123 to stabilize their position in the pocket. These two residues have been reported

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to participate in the formation of hydrogen bond network to stabilize the activation

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function-2 (AF-2) helix (Dhoke et al., 2012), which is further confirmed in this work. Moreover, hydrogen bonds that exist between the carboxylate group and other residues (such as Phe82 and Phe160) can also help to enhance the receptor-ligand

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binding affinity.

As shown in Table 2, the calculated binding energies of bisphenols with

mPPARα-LBD* rank as BPC > HPTE > BPA > BPAF, TBBPA > triBBPA > diBBPA > monoBBPA > BPA and TCBPA > triCBPA > diCBPA > monoCBPA > BPA

respectively when different halogenation patterns were analyzed. In the case of the compounds with the halogen substituents on the phenolic rings, the binding energies increase with the increase of halogen atoms (Fig. 4A and B), which is consistent with previously reported work that the activation of hPPARγ depended on the halogenation

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degree of bisphenol analogues. The bulkier halogenated BPAs, the greater their capability to activate hPPARγ (Riu et al., 2011b). Compared to the higher congeners,

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the weaker affinity of the lower halogenated BPAs with mPPARα-LBD* might be explained by their smaller size and less direct contacts with the receptor. In addition,

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BPC, HPTE, and BPAF with the halogens on the bridging alkyl moiety also show

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distinct receptor-ligand affinity, in which BPC and HPTE have more negative binding

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energies than BPA while BPAF shows less negative energy. This difference might be due to the steric effect of substituents, which plays a role in receptor-ligand binding,

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especially for the congeners with higher halogen numbers.

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In conclusion, the results of molecular docking showed that the halogenated BPAs

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exhibited different interaction modes and binding energies to mPPARα-LBD*, mainly due to the distinct halogenation patterns on the bridge methyl groups and the phenol rings. Additionally, the linear relationship (R2 = 0.6659) between the docking scores

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and Kd values of the halogenated BPAs is shown in Fig. 4C, indicating that this study may be helpful for predicting the toxic potency of novel bisphenol analogues. 4. Conclusion In this work, the fluorescence polarization assay together with molecular dynamics

simulations was utilized to investigate the binding of halogenated BPAs toward mPPARα-LBD*, facilitating the elucidation of molecular recognition mechanism. The in vitro competitive binding assay showed that halogenated BPAs are affinity ligands for mPPARα-LBD*. The electron withdrawing effect of the halogens disturbs the

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charge distribution and leads to a change in the electron cloud around these

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compounds. The variation of electronic properties was illustrated with a dependence

on the halogenation patterns. Furthermore, a mechanistic study on the interactions between halogenated BPAs and mPPARα-LBD* at atomic level was performed.

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Interestingly, a good correlation was observed between the simulated binding energies

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and experimental Kd values of halogenated BPAs. These results not only provide

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valuable information for better understanding of the binding modes between halogenated BPAs and mPPARα-LBD*, but are also helpful for the production of

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Conflict of interest

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eco-friendly materials with reduced toxicities.

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The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the National Key Research and Development Program

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of China (2017YFD0300303), the National Natural Science Foundation of China (31601534), and the Agricultural Science and Technology Innovation Program of Jilin Province (CXGC2017JQ006 and CXGC2017JQ010). Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. References

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chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878, 3363-3369. Kilroy, G.E., Zhang, X., Floyd, Z.E., 2009. PPARγ AF-2 domain functions as a component of a ubiquitin-dependent degradation signal. Obesity (Silver Spring) 17, 665-673. Kitamura, S., Suzuki, T., Sanoh, S., Kohta, R., Jinno, N., Sugihara, K., Yoshihara, S.i., Fujimoto, N., Watanabe, H., Ohta, S., 2005. Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds. Toxicol. Sci. 84, 249-259. Krey, G., Braissant, O., L’Horset, F., Kalkhoven, E., Perroud, M., Parker, M.G., Wahli, W., 1997. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay. Mol. Endocrinol. 11, 779-791. Matthews, J.B., Twomey, K., Zacharewski, T.R., 2001. In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors α and β. Chem. Res. Toxicol. 14, 149-157. Mayerhöffer, U., Fimmel, B., Würthner, F., 2012. Bright near-infrared fluorophores based on squaraines by unexpected halogen effects. Angew. Chem. Int. Ed. Engl. 51, 164-167. Moriyama, K., Tagami, T., Akamizu, T., Usui, T., Saijo, M., Kanamoto, N., Hataya, Y., Shimatsu, A., Kuzuya, H., Nakao, K., 2002. Thyroid hormone action is disrupted by bisphenol A as an antagonist. J. Clin. Endocrinol. Metab. 87, 5185-5190. Paris, F., Balaguer, P., Térouanne, B., Servant, N., Lacoste, C., Cravedi, J.-P., Nicolas, J.-C., Sultan, C., 2002. Phenylphenols, biphenols, bisphenol-A and 4-tert-octylphenol exhibit α and β estrogen activities and antiandrogen activity in reporter cell lines. Mol. Cell. Endocrinol. 193, 43-49. Riu, A., Grimaldi, M., le Maire, A., Bey, G., Phillips, K., Boulahtouf, A., Perdu, E., Zalko, D., Bourguet, W., Balaguer, P., 2011a. Peroxisome proliferator-activated receptor γ is a target for halogenated analogs of bisphenol A. Environ. Health Perspect. 119, 1227-1232. Riu, A., le Maire, A., Grimaldi, M., Audebert, M., Hillenweck, A., Bourguet, W., Balaguer, P., Zalko, D., 2011b. Characterization of novel ligands of ERα, Erβ, and PPARγ: the case of halogenated bisphenol A and their conjugated metabolites. Toxicol. Sci. 122, 372-382. Rubin, B.S., 2011. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. J. Steroid Biochem. Mol. Biol. 127, 27-34. Sui, Y., Ai, N., Park, S.-H., Rios-Pilier, J., Perkins, J.T., Welsh, W.J., Zhou, C., 2012. Bisphenol A and its analogues activate human pregnane X receptor. Environ. Health Perspect. 120, 399-405. Sun, H., Wang, L., Zhou, Q., 2013. Effects of bisphenol A on growth and nitrogen nutrition of roots of soybean seedlings. Environ. Toxicol. Chem. 32, 174-180. Takayanagi, S., Tokunaga, T., Liu, X., Okada, H., Matsushima, A., Shimohigashi, Y.,

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IP T SC R U

A

N

Fig. 1. Competitive binding of halogenated BPAs to mPPARα-LBD*. Results are

A

CC E

PT

ED

M

given as means ± SEM of three independent experiments.

HPTE

BPAF

TBBPA

diBBPA

triBBPA

monoCBPA

diCBPA

triCBPA

N

TCBPA

A

-0.02

U

monoBBPA

IP T

BPC

SC R

BPA

0.02

M

Fig. 2. Electrostatic potential (ESP) maps of halogenated BPAs. The electronegative

A

CC E

PT

ED

and electropositive ESP surfaces are colored in red and blue, respectively.

IP T SC R U N

A

Fig. 3. Computational docking of halogenated BPAs to mPPARα-LBD*. Top: the

M

hydrophobic binding pocket of mPPARα-LBD* to stabilize BPAF(A) and TBBPA(B), respectively. Bottom: the profiles of BPAF(C)/TBBPA(D)-mPPARα-LBD* interaction,

ED

showing the hydrogen bonds formed between ligands and amino acid residues of the

A

CC E

PT

receptor.

IP T SC R U N A M ED PT

CC E

Fig. 4. Correlation of the calculated binding energies to the number of halogen atoms

A

(A and B) and the experimental binding affinities (C) for halogenated BPAs.

Table 1

BPA

12.27

10.80

BPC

13.77

12.12

HPTE

12.60

11.09

BPAF

20.34

17.90

monoBBPA

15.32

13.48

diBBPA

11.95

10.52

triBBPA

12.25

10.78

TBBPA

8.58

7.55

monoCBPA

13.69

12.05

diCBPA

12.81

triCBPA

14.30

TCBPA

12.72

A 11.27

12.58

M

ED PT CC E A

SC R

Kd (μM)

U

IC50 (μM)

N

Compound

IP T

IC50 values and dissociation constants (Kd) of halogenated BPAs.

11.19

Table 2 The molecular length, Connolly solvent-excluded volume (CSEV), and docking results of halogenated BPAs to mPPARα-LBD*. Length

CSEV

Binding energy

(Å)

(Å3)

(kcal mol-1)

BPA

9.99

205.5

-6.22

Tyr123

BPC

10.49

212.1

-6.74

Tyr123

HPTE

10.03

230.0

-6.32

BPAF

10.10

230.7

-4.12

monoBBPA

9.39

225.8

-6.95

diBBPA

9.39

245.8

-7.48

triBBPA

10.69

265.9

TBBPA

10.69

286.0

monoCBPA

9.37

diCBPA

Phe160

N

U

Phe160

Ser89, Tyr123 Ser89, Tyr123 Ser89, Tyr123

-8.08

Phe82

220.2

-6.79

Ser89, Tyr123

10.41

234.5

-7.42

Ser89, Tyr123

triCBPA

9.91

248.9

-7.52

Ser89, Tyr123

TCBPA

10.41

263.6

-7.99

Phe82

M

ED

PT

A

-7.88

CC E A

IP T

Hydrogen bonds

SC R

Compound