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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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Environmental Science & Technology Letters

1

Organophosphate Esters Bind to and Inhibit Estrogen Related

2

Receptor γ in Cells

3 4

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

5 6

1

7

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

10 11 12

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

13

Environmental

14

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

15

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

16

[email protected]

Chemistry

and

Eco-toxicology,

17

1

ACS Paragon Plus Environment

Research

Center

for


18

ABSTRACT

19

Organophosphate esters (OPEs) have been reported to induce endocrine

20

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

21

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

22

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

25

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

26

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

27

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

29

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

30

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

31

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

32

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

33

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

34

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

35

disruption effects of OPEs.

36 37 38

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

39

nuclear receptors, toxicological mechanism, endocrine disruption effect

40 2


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41

INTRODUCTION

42

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

43

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

44

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

45

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

46

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

47

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

48

in experimental animals4, 5 and humans6, 7.

49

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

50

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

51

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

52

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

53

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

54

hydrocarbon receptor10, peroxisome proliferator-activated receptor11 and thyroid

55

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

56

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

57

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

58

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

59

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

60

ERRγ can affect the estrogenic responses through interplaying with ERs signaling

61

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

62

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.

64

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

65

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

66

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

67

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

68

aroused in environmental toxicology studies in the past few years19,

69

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

70

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

71

signaling pathways at nanomolar to micromolar levels19, 20.

20

. Some

72

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

73

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

74

OPEs with different substitution groups with ERRγ by a fluorescence competitive

75

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

76

mediated luciferase reporter gene assay. Molecular docking analysis was then

77

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

78

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

79

Materials and Methods

80

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

81

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

82

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

83

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

84

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


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85

phosphate

(TPhP),

2-ethylhexyl

diphenyl

phosphate

(EHDPP),

86

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

87

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

88

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

89

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

90

tested compounds are shown in Figure S1.

91

Fluorescence competitive binding assay. The binding potency of OPEs with

92

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

93

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

94

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

95

S2–S7).

96

ERRγ Mediated Luciferase Reporter Gene Assay. ERRγ expression plasmid

97

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

98

three copies of ERRE (5’-CCGGACCTCAAGGTCACGTTCGGACCTCAAGGTCA

99

CGTTCGGACCTCAAGGTCAGGATCCA-3’) were constructed by GeneChem

100

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

101

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

102

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

103

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

104

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

105

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

106

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



107

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

108

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

109

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

110

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

111

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

112

supporting information.

113

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

114

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

115

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

116

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

117

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

118

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

119

same as described in a previous publication23.

120

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

121

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

122

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

147

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

148

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

149

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

150

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



151

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

152

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

153

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

154

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

155

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

156

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

157

of the substitution groups.

158

Activity of OPEs towards ERRγ. Different from some common nuclear

159

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

160

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

161

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

162

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

163

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

164

ERRγ transcriptional activity while inverse agonist 4-OHT displayed inhibitory

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

174

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

175

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

176

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

177

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

178

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

179

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

180

and Figure S10).

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

Molecular docking of the interaction of OPEs with ERRγ. Molecular

193

docking analysis was performed between OPEs and ERRγ to provide some

194

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



195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

28

, indicating the feasibility of the docking method.

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

disturbing the AF-2 helix from the active position.

236 237 238

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



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239 240

Acknowledgments

241

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

242

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

243

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

244

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

249

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.

251 252

12


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254

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349

17



350

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.

18


Page 18 of 22

Page 19 of 22


355 356

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

366

determined by ChemBioDraw.

367

19



368 369

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)

372

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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381 382

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

383

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



393

For Table of Contents Only

394 395

Manuscript title: Organophosphate Esters Bind to and Inhibit Estrogen Related

396

Receptor γ in Cells

397

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

22


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Organophosphate Esters Bind to and Inhibit ... - ACS Publications

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