Organophosphate Esters Bind to and Inhibit Estrogen-Related

Jan 25, 2018 - Organophosphate Esters Bind to and Inhibit Estrogen-Related. Receptor γ in Cells. Lin-Ying Cao,. †,‡. Xiao-Min Ren,*,†. Chuan-Ha...
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Organophosphate Esters Bind to and Inhibit Estrogen Related Receptor # in Cells Lin-Ying Cao, Xiao-Min Ren, Chuan-Hai Li, and Liang-Hong Guo Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.7b00558 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Organophosphate Esters Bind to and Inhibit Estrogen Related

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Receptor γ in Cells

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Lin-Ying Cao1,2, Xiao-Min Ren1*, Chuan-Hai Li1,2, Liang-Hong Guo1,2*

5 6

1

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Center for Eco-environmental Sciences, Chinese Academy of Sciences, 18

8

Shuangqing Road, P.O. Box 2871, Beijing 100085, China

9

2

State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research

University of Chinese Academy of Sciences, Beijing 100039, P. R. China

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*Address correspondence to: Prof. Liang-Hong Guo, State Key Laboratory of

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Environmental

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Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road,

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P.O. Box 2871, Beijing 100085, P. R. China. Telephone/Fax: 86 010 62849685. e-mail:

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[email protected]

Chemistry

and

Eco-toxicology,

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ABSTRACT

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Organophosphate esters (OPEs) have been reported to induce endocrine

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disruption effects, and several well known nuclear receptors have been investigated as

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cellular targets of OPEs in their mode of action. Here, we demonstrated for the first

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time that an orphan nuclear receptor estrogen-related receptor γ (ERRγ) is another

23

possible target of OPEs. Using the fluorescence competitive binding assays

24

established by ourselves, we measured the binding affinity of 9 OPEs with different

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substitution groups including aromatic rings, chlorinated alkyl chains and alkyl chains.

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Seven of the OPEs were found to bind to ERRγ, with tri-m-cresyl phosphate (TCrP)

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showing the highest binding affinity (Kd, 0.34 µM). By using an ERRγ mediated

28

luciferase reporter gene assay, we found the seven OPEs showed inhibitory effects

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towards ERRγ. Both the binding affinity and inhibitory effect of the OPEs correlate

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positively with the hydrophobicity of their substitution groups in the rank order of

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aromatic rings > chlorinated alkyl chains > alkyl chains. Based on the molecular

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docking, the mechanism of the inhibitory effect of OPEs was proposed to be

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ligand-triggered displacement of activation function-2 helix from the active position

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in the receptor. ERRγ pathway may provide a new mechanism for the endocrine

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disruption effects of OPEs.

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Key words: estrogen-related receptor γ (ERRγ), organophosphate esters (OPEs),

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nuclear receptors, toxicological mechanism, endocrine disruption effect

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INTRODUCTION

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Organophosphate esters (OPEs) are the esters of phosphoric acid with different

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substitution groups such as aromatic rings, halogenated alkyl chains and alkyl chains1.

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Currently, OPEs have been widely used in industrial products, such as textiles,

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furniture, polyurethane foam, electronics and paints2. As a result, OPEs have been

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detected in various environmental media and human samples3. Numerous studies on

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the toxicology of OPEs have showed their disruption effects on the endocrine system

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in experimental animals4, 5 and humans6, 7.

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Up to now, many in vitro studies have been performed to reveal the molecular

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mechanisms of the endocrine disruption effects of OPEs. Based on these mechanistic

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studies, the OPEs might exert endocrine disruption effects by disturbing signaling

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pathways mediated by some nuclear receptors, such as estrogen receptors (ERs),

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androgen receptor8,9, pregnane X receptor8,9, glucocorticoid receptor9, aryl

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hydrocarbon receptor10, peroxisome proliferator-activated receptor11 and thyroid

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hormone receptor5, 9. However, the mode of action for the endocrine disruption effects

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of OPEs through these nuclear receptor mediated pathways has not been established.

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Estrogen-related receptor γ (ERRγ) is one of estrogen-related receptors (ERRs),

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which consists of three isoforms (ERRα, ERRβ and ERRγ)12. ERRγ has been proved

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to play important roles in many human endocrine-related functions. For example,

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ERRγ can affect the estrogenic responses through interplaying with ERs signaling

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pathways by sharing the co-regulators or regulating the same target genes12. In

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addition, ERRγ can regulate steroidogenesis in testicles by regulating steroidogenic 3

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gene expression, which could also influence the endocrine-related functions13.

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Besides, ERRγ has been found to be highly expressed in some hormone-related

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cancers and has been regarded as a potential biomarker and drug target for the

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treatment of breast cancer14, 15, ovarian cancer16, 17 as well as prostate cancer18. Due to

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the important physiological and pathological functions of ERRγ, attentions have been

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aroused in environmental toxicology studies in the past few years19,

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environmental compounds, such as bisphenol A (BPA), BPA analogues and

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alkylphenols, have been reported to bind with ERRγ and affect the subsequent

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signaling pathways at nanomolar to micromolar levels19, 20.

20

. Some

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In the present study, we aimed to find out whether ERRγ could be another

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potential target of OPEs in cells. We first investigated the binding interaction of 9

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OPEs with different substitution groups with ERRγ by a fluorescence competitive

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binding assay. We further investigated their activities towards ERRγ by receptor

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mediated luciferase reporter gene assay. Molecular docking analysis was then

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performed to simulate the interactions of these compounds with the receptor in order

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to investigate the structural basis of their binding and activity with ERRγ.

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Materials and Methods

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Chemicals. Human ERRα, ERRβ and ERRγ ligand binding domains (LBDs)

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were purchased from Invitrogen (Carlsbad, CA, USA). GSK4716, 4-OHT and

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17β-estradiol (E2) (all with purity ≥ 98%) were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). Fluorescein isothiocyanate (FITC, 98%) was purchased from

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Merck (KGaA, Darmstadt, Germany). Tri-m-cresyl phosphate (TCrP), triphenyl 4

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phosphate

(TPhP),

2-ethylhexyl

diphenyl

phosphate

(EHDPP),

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tris(1,3-dichloro-2-propyl) phosphate (TDCP), tris (2-chloroisopropyl) phosphate

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(TCPP), tri(2-chloroethyl) phosphate (TCEP), tri-n-butyl phosphate (TnBP), triethyl

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phosphate (TEP) and trimethyl phosphate (TMP) (all with purity ≥ 98%) were

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purchased from Dr. Ehrenstorfer Gmbh (Augsburg, Germany). The structures of the

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tested compounds are shown in Figure S1.

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Fluorescence competitive binding assay. The binding potency of OPEs with

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ERRγ was determined by fluorescence polarization (FP) based competitive binding

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assay using GSK-FITC as the site-specific fluorescence probe. The establishment and

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validation of the method were described in supporting information in detail (Figures

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S2–S7).

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ERRγ Mediated Luciferase Reporter Gene Assay. ERRγ expression plasmid

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(pcDNA3/ERR-γ), firefly luciferase reporter plasmid (pGL3/3×ERRE), containing

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three copies of ERRE (5’-CCGGACCTCAAGGTCACGTTCGGACCTCAAGGTCA

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CGTTCGGACCTCAAGGTCAGGATCCA-3’) were constructed by GeneChem

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(Shanghai, China). Renilla luciferase reporter plasmid pRL-TK was purchased from

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Promega (Madison, WI, USA) and used as an internal control. HeLa cells (2×104

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cells/well) were seeded in a 96-well plate and incubated for 24 h. Then the cells in

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each well were transfected with 35 ng pcDNA3/ERR-γ, 35 ng pGL3/3×ERRE and 35

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ng pRL-TK using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s

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protocol. After 24 h, the cells were exposed to different concentrations of the tested

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compound in phenol red-free DMEM for another 24 h. The concentrations of OPEs 5

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used in the experiment were all non-cytotoxic as determined by WST-1 assay (Figure

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S9). Finally, both firefly and renilla luciferase activities were measured using a

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Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The ERRγ

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transcriptional activity was expressed as the ratio of firefly luciferase signal to renilla

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luciferase signal. The details for the cell culture and WST-1 assay were showed in the

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supporting information.

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Molecular Docking Analysis. The 3D crystal structure of ERRγ-LBD (bound to

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an inverse agonist 4-OHT, PDB ID: 2GPU) was obtained from the Protein Data Bank

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(http://www.rcsb.org/pdb)21. The PDB format of ligands with 3D-coordinates were

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obtained by the PRODRG server22. AutoDock 4.2 (Scripps Research Institute, CA,

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USA), employing a Lamarckian genetic algorithm for the conformational search, was

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used to determine the interaction of ligands with ERRγ. The details of docking are the

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same as described in a previous publication23.

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Statistical Analysis. All the experiments were repeated three times and the

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results were expressed by mean ± SD (n=3, SD means the standard deviation of 3

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measurements). Statistical analysis was performed using Student’s t-test. A p-value
TPhP, EHDPP > TDCP, TCPP > TCEP, TnBP > TEP,

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TMP (Figure 1B). The general trend could be found that the binding potency of OPEs

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with aromatic rings substitution > chlorinated alkyl chains substitution> alkyl chains

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substitution. Secondly, we found the ERRγ binding potency of OPEs is in positive

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correlation with their hydrophobicity (LogKow), as indicated by the good positive 7

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linear relationship (R2 = 0.90) between LogKow and binding potency (Figure 1C). The

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hydrophobicity (LogKow) of the substitution groups is in the order of aromatic rings >

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chlorinated alkyl chains > alkyl chains (Table 1). Since the hydrophobicity of OPEs is

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determined by the substitution groups, the group with higher hydrophobicity leads to

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stronger ERRγ binding potency. Overall, we provided the first evidence that OPEs

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could bind to ERRγ, and their binding potency is closely related to the hydrophobicity

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of the substitution groups.

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Activity of OPEs towards ERRγ. Different from some common nuclear

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receptors like ER and TR for which the transcriptional activity is stimulated by ligand

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binding, ERRγ is a self-activated nuclear receptor showing constitutive basal

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transcriptional activity without any ligand stimulation24, 25. Binding of an agonist to

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ERRγ enhances the transcriptional activity, while an inverse agonist inhibits the

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transcriptional activity26, 27. As shown in Figure S10, agonist GSK4716 increased the

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ERRγ transcriptional activity while inverse agonist 4-OHT displayed inhibitory

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effects in a dose-dependent manner with the lowest effective concentration (LOEC) of

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100 nM and 1 µM respectively. These results are in good agreement with the previous

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studies (LOEC of about 100 nM and 1 µM for GSK4716 and 4-OHT respectively)26, 27,

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confirming the reliability of the ERRγ-mediated luciferase reporter gene assay.

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For the seven OPEs could bind to ERRγ, they showed inhibitory effects on the

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ERRγ transcriptional activity, indicating they were inverse agonists for ERRγ. Three

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aromatic ring substituted OPEs (TCrP, TPhP and EHDPP) inhibited the ERRγ

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transcriptional activity in a dose-dependent manner with the LOEC of 1 µM, 10 µM 8

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and 10 µM and the maximum inhibitory rate of 64%, 46% and 59%, respectively

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(Figure 2A and Figure S10). For the three chlorinated alkyl chain substituted OPEs

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(TDCP, TCPP and TCEP), they showed inhibitory effect with the LOEC of 10 µM, 10

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µM and 100 µM, and the maximum inhibitory rate of 44%, 42% and 30%,

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respectively (Figure 2A and Figure S10). For the three alkyl chain substituted OPEs,

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only TnBP inhibited the transcriptional activity with the LOEC of 10 µM and the

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maximum inhibitory rate of 30%, while TEP and TMP showed no effect (Figure 2A

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and Figure S10).

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We further compared the inhibitory effects of these 9 OPEs at the highest tested

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concentration (100 µM). The rank order of the 9 OPEs is TCrP, EHDPP > TPhP >

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TDCP, TCPP > TCEP, TnBP > TEP, TMP (Figure 2B). It is obvious that this order is

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very similar to the order of the binding potency of the 9 OPEs. Actually, there exists a

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close positive correlation (R2=0.98) between the inhibition rate of OPEs and their

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binding potency with ERRγ-LBD (Figure 2C). This correlation suggests that the

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ERRγ inhibitory effect of an OPE is probably dictated by its binding potency with

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ERRγ. As shown in Figure 2D, there also exists a close positive correlation (R2=0.98)

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between the inhibitory effect and LogKow of the 9 OPEs. The combined results of

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competitive binding assay and reporter gene assay indicate that both the binding

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potency and inhibitory effects of OPEs are closely related to their hydrophobicity.

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Molecular docking of the interaction of OPEs with ERRγ. Molecular

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docking analysis was performed between OPEs and ERRγ to provide some

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explanations about the structural characteristics of their binding and activity with 9

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ERRγ. Previous crystal structure studies have revealed the ligand binding pocket of

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ERRγ is composed of several hydrophobic and polar residues from helix (H)3, H5,

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H10 and activation function-2 (AF-2) helix21. Besides, the pocket can be divided into

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two parts by residues Arg316 and Asp275 serving as gatekeepers28. As shown in

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Figure 3A, 4-OHT resides in the outside part of the pocket near AF-2 helix, forming

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hydrogen bonds with Glu275, Arg316 and Asp273. The docking results for 4-OHT are

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in line with the results obtained from the crystal structures of ERRγ/4-OHT complex21,

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, indicating the feasibility of the docking method.

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For the 9 tested OPEs, all of them could fit into the ligand binding pocket of

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ERRγ but with different binding energies (Figure 3B-D, Figure S11 and Table 1). For

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Autodock, the higher binding energy means lower binding potency with the protein.

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By comparing the calculated △G value (Table 1) and the measured binding potency,

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we found there exists a close correlation (R2=0.88) between each other (Figure 3E).

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As shown in Figure 3B-D and Figure S11, the 9 OPEs bound to ERRγ at the same

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binding site as 4-OHT. However, different from 4-OHT, no hydrogen bond was

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formed between OPEs and ERRγ (Table 1). So we speculate the binding potency of

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OPEs is probably dominated by their hydrophobic interactions with ERRγ, which was

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dependent on the hydrophobicity of substitution groups. The good linear relationship

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(R2=0.82) between △G value and LogKow of the 9 OPEs supports our speculation

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(Figure 3F). Overall, our binding and docking analysis results strongly suggest that

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hydrophobic interactions play important roles in the binding of OPEs to ERRγ.

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The crystal structures of agonistic and inverse agonistic conformation of ERRγ 10

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revealed that the position of C-terminal AF-2 helix plays an important role in the

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receptor activation21. The crystal structure of ERRγ/4-OHT complex suggests the

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inhibitory effect of 4-OHT is probable due to the steric hindrance of the benzene ring

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and dimethylaminoethoxy-phenyl, both of which could result in relocation of AF-2

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away from the active position capping the ligand binding pocket21. By analyzing the

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binding mode of 7 OPEs exerting inhibitory activity, we found they displayed a

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binding geometry very similar to that of 4-OHT, with two substitution group

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(aromatic ring, chlorinated alkyl chain or alkyl chain) at the same site as the benzene

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ring and dimethylaminoethoxy-phenyl of 4-OHT, respectively (Figure 3B-D and

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Figure S11). These substitution groups of OPEs might lead to relocation of AF-2 helix

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from the active position in the same manner as 4-OHT. Therefore, we speculate the

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OPEs might exert inhibitory effect in a mechanism similar to 4-OHT, which is the

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ligand-induced relocation of AF-2 helix from the active position.

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To conclude, we found ERRγ is another potential target of OPEs in cells for their

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endocrine disruption effects. OPEs could bind to ERRγ and inhibit its transcriptional

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activity. Both the binding potency and inhibitory effect of OPEs towards ERRγ are

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closely related to the hydrophobicity of their substitution groups. Based on the

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molecular docking analysis, the OPEs might exert their inhibitory effects by

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disturbing the AF-2 helix from the active position.

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Notes The authors declare that there are no conflicts of interest. 11

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Acknowledgments

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This work was supported by the Chinese Academy of Sciences (XDB14040100,

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QYZDJ-SSW-DQC020), the National Natural Science Foundation of China

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(91543203, 21621064, and 21777187) and the Royal Society International

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Collaboration Award for Research Professors (IC160121).

245 246 247

Supporting Information Synthesis

and

characterization

of

fluorescence

probe

GSK-FITC,

the

248

establishment and validation of the competitive binding method, the cytotoxicity of

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OPEs on HeLa cells, molecular docking of 4-OHT and OPEs with ERRγ. This

250

material is available free of charge via the Internet at http://pubs.acs.org.

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ERR gamma. J. Med. Chem. 2005, 48, 3107-3109.

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(27) Coward, P.; Lee, D.; Hull, M. V.; Lehmann, J. M. 4-hydroxytamoxifen binds to

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and deactivates the estrogen-related receptor gamma. Proc. Natl. Acad. Sci. U. S. A.

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2001, 98, 8880-8884.

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(28) Kim, Y.; Koh, M.; Kim, D. K.; Choi, H. S.; Park, S. B. Efficient discovery of

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selective small molecule agonists of estrogen-related receptor gamma using

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combinatorial approach. J. Comb. Chem. 2009, 11, 928-937.

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Table 1. IC50, Kd, LogKow and docking results of 4-OHT and 9 OPEs with ERRγ. Compounds

IC50

Kd

LogKow

(µM)

(µM)

4-OHT

0.04

0.043

5.7

-12.7

TCrP

0.31

0.34

7.0

-9.5

TPhP

2.60

2.83

5.6

-8.6

EHDPP

1.30

1.42

6.7

-8.9

TDCP

NA

NA

5.0

-5.7

TCPP

NA

NA

4.0

-5.4

TCEP

NA

NA

2.7

-3.9

TnBP

NA

NA

4.2

-4.7

TEP

ND

ND

1.6

-3.6

TMP

ND

ND

-0.6

-3.1

△G (kcal/mol)

351

NA, Not available (a compound could displace the fluorescence probe from

352

ERRγ-LBD but could not obtain the IC50 value). ND, Not determined (a compound

353

could not displace the fluorescence probe from ERRγ-LBD even at the highest tested

354

concentration). The logKow values for the chemicals were obtained by ChemBioDraw.

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Figure 1. The competitive binding curves of 9 OPEs to ERRγ-LBD and the

357

relationship between binding potency and LogKow of these OPEs. (A) The competitive

358

binding curves of 3 representative OPEs to ERRγ-LBD. The competitive binding

359

curves were plotted the relative FP values of 50 nM GSK-FITC at 520 nm in presence

360

of 200 nM ERRγ-LBD as a function of concentrations of tested compounds. (B)

361

Comparison of the binding potency of 9 OPEs with ERRγ-LBD by using the

362

percentage of probe displacement at 0.5 µM. (C) The linear relationship between

363

binding potency with ERRγ-LBD (percentage of probe displacement at 0.5 µM) and

364

LogKow of OPEs. The percentage of competitive binding potency of control group

365

(Ctrl, 0.1% DMSO) was set as 0%. The logKow values for the chemicals were

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determined by ChemBioDraw.

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Figure 2. Activity of 9 OPEs towards ERRγ and the relationships between the

370

inhibitory effect and binding potency as well as LogKow of OPEs. (A) Effects of 3

371

representative OPEs on ERRγ-mediated luciferase transcriptional activity. (B)

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Comparison of the inhibitory effect of 9 OPEs towards ERRγ by using the luciferase

373

transcriptional inhibitory activity determined at 100 µM. (C) The linear relationship

374

between the inhibitory effect of OPEs on ERRγ (luciferase transcriptional inhibitory

375

activity determined at 100 µM) and binding potency of OPEs to ERRγ (percentage of

376

probe displacement at 0.5 µM). (D) The linear relationship between the inhibitory

377

effect of OPEs on ERRγ (luciferase transcriptional inhibitory activity determined at

378

100 µM) and LogKow of OPEs. The percentage of inhibition of control group (Ctrl,

379

0.1% DMSO) was set as 0%. *p < 0.05, compared with the Ctrl group.

380

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Figure 3. The binding pose of 4-OHT and OPEs in ERRγ obtained by molecular

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docking and the relationships between △G values and binding potency as well as

384

LogKow of OPEs. (A) The binding pose of 4-OHT in ERRγ. ERRγ was shown as blue

385

ribbons and 4-OHT was shown in sticks; the H3, H5, H10 and AF-2 helix consisting

386

of the ligand binding pocket were labeled; four important residues were also labeled.

387

(B)-(D) Overlay of the binding pose of TCrP (B), TDCP (C) and TnBP (D) with

388

4-OHT. 4-OHT was shown in green sticks and OPEs were shown in gray sticks

389

[carbon (C), gray; oxygen (O), red; chlorine (Cl), green; phosphorus (P), yellow]. (E)

390

The linear relationship between △G values and binding potency of OPEs (percentage

391

of probe displacement at 0.5 µM) to ERRγ. (F) The linear relationship between △G

392

values and LogKow of OPEs. 21

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For Table of Contents Only

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Manuscript title: Organophosphate Esters Bind to and Inhibit Estrogen Related

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Receptor γ in Cells

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Authors: Lin-Ying Cao, Xiao-Min Ren, Chuan-Hai Li, Liang-Hong Guo

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