MeO-PBDEs on Androgen Receptor: In Vitro

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Effects of HO-/MeO-PBDEs on Androgen Receptor: In Vitro Investigation and Helix 12-Involved MD Simulation Xiaoxiang Wang, Huaiyu Yang, Xinxin Hu, Xiaowei Zhang, Qiansen Zhang, Hualiang Jiang, Wei Shi, and hongxia yu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es4029364 • Publication Date (Web): 18 Sep 2013 Downloaded from http://pubs.acs.org on September 23, 2013

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Environmental Science & Technology

1

Effects of HO-/MeO-PBDEs on Androgen Receptor:

2

In Vitro Investigation and Helix 12-Involved MD Simulation

3 4

‡ † † ‡ Xiaoxiang Wang †, Huaiyu Yang , Xinxin Hu , Xiaowei Zhang , Qiansen Zhang ,

5

Hualiang Jiang ‡,

Wei Shi

†,*

and Hongxia Yu

†,*

6 7 8 9 10

†

State Key Laboratory of Pollution Control and Resources Reuse, School of the

Environment, Nanjing University, Nanjing 210023, PR China, ‡

Drug Discovery and Design Center, State Key Laboratory of Drug Research,

11

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi

12

Road, Shanghai 201203, PR China.

13 14 15 16 17 18 19 20 21 22

Submitted to: Environmental Science and Technology

ACS Paragon Plus Environment


23

ABSTRACT

24

Hydroxylated and methoxylated polybrominated diphenyl ethers (HO-/MeO-PBDEs) have

25

received increasing attention for their potential endocrine disrupting activities and widely

26

environmental distribution. However, little information is available for the anti-androgenic

27

activities, and the molecular mechanism of interactions with androgen receptor (AR) was not fully

28

understood. In the present study, cell line assay and computational simulation were integrated to

29

explore the molecular mechanism of interactions between chemicals and AR systematically. The

30

metabolites with similar molecular structures exhibited different anti-androgenic activities while

31

none of them showed androgenic activities. According to the multi-system molecular dynamics

32

simulation minute differences in the structure of ligands induced dramatic different

33

conformational transition of AR-ligand binding domain (LBD). The Helix12 (H12) component of

34

active ligands occupied AR-LBD could become stable, but this component continued to fluctuate

35

in inactive ligands occupied AR-LBD. Settling time and reposition of H12 obtained in dynamics

36

process are important factors governing anti-androgenic activities. The related settling times were

37

characteristic for anti-androgenic potencies of the tested chemicals. Overall, in our study, the

38

stable reposition of H12 is characterized as a computational mark for identifying AR antagonists

39

from PBDE metabolites, or even other various environmental pollutants.

40 41 42 43 44


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INTRODUCTION

46

As brominated flame retardants, polybrominated diphenyl ethers (PBDEs) are used as additives in

47

a variety of household and industrial products. Due to their persistence and potential for

48

bio-accumulating1, PBDEs have received substantial attention from the perspective of

49

environment2-4. Some studies suggest that the toxicity of PBDEs might be due in part to their

50

metabolites5, 6. Most recently, their environmental metabolites, i.e., hydroxylated PBDEs

51

(HO-PBDEs) and methoxylated PBDEs (MeO-PBDEs), have been widely observed in human

52

blood

53

wildlife

54

chemicals (EDCs)

55

thyroid receptor (TR), estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) 13-15.

6, 7

, paired maternal and cord sera 8, and breast milk 9, as well as in marine and terrestrial

10, 11

. Some of these PBDE metabolites are classified as potential endocrine disrupting 12

due to the reported potential endocrine disrupting activities mediated by the

56

Kojima et al. have reported some HO-PBDEs and MeO-PBDEs could inhibit the androgen

57

receptor (AR) activity 16. Additional studies have detected the potential anti-androgenic activity of

58

HO-PBDEs and MeO-PBDEs using reporter gene assays, and the metabolites may possess higher

59

activity than the precursors

60

indicated that their activities vary widely. However, limited information is available for the

61

mechanisms to explain this phenomenon. Neither the mark for screening active chemicals from

62

numerous analogs nor the factors that determine potency have been explicated exhaustively. In

63

addition, detecting AR-related activity of the structural diverse metabolites in vivo and in vitro is

64

time-consuming and difficult to achieve. Therefore, it is important to understand the mechanism of

65

the ligand-AR interaction and to develop methods for predicting the anti-/androgenic activity of

66

HO-PBDEs and MeO-PBDEs. Interests in developing computational methods to investigate the

17, 18

. The structures of these chemicals are similar, but the results



67

mechanism of ligands binding AR and predict the anti-/androgenic activity are increasing greatly

68

19

69

using of them have been performed to provide insights into the ligand-receptor interaction and to

70

predict biological activity20-22. However, QSAR models rely on large amounts of training data,

71

existing data for AR activities of HO-PBDEs and MeO-PBDEs is far less enough. Meanwhile, the

72

inflexibility of the backbone or even all atoms of the receptor in molecular docking makes the

73

ligand-receptor binding extremely uncertain.

. Quantitative structure-activity relationship (QSAR), molecular docking and combinational

74

Previous studies have revealed that the reposition of Helix12 (H12) in ligand binding domain

75

(LBD) of nuclear receptors (NRs) plays a key role in the function of NRs in pharmacology23-25.

76

Considering that the AR is a member of NR superfamily, exploring the effect of H12 repositioning

77

on the anti-/androgenic activity of HO-PBDEs and MeO-PBDEs could enhance the understanding

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of binding and activating mechanism. Molecular dynamics (MD) simulation has been applied to

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explore the dynamic reposition of H12 and validate the significance previously 26-28. This method

80

gives a view of the motion, which could avoid the disadvantages of QSAR and molecular docking.

81

In this study, a cell-based assay was performed to detect the anti-androgenic activity of

82

HO-/MeO-PBDEs. An unrestrained all-atom MD simulation and other computational methods

83

were combined to provide insights into how the metabolites of PBDEs bind to and activate the AR

84

from the conformational transitional perspective. The mark for screening active chemicals and the

85

decisive factor in determining potency were both characterized. Based on these results, a

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computational server for predicting the AR binding and biological properties of HO-PBDEs and

87

MeO-PBDEs was built for public service. The methodology of this research might be beneficial

88

for discovering marks for screening anti-/androgenic chemicals from other classes of chemicals


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and the environmental EDCs mediated by other receptors.

90 91

MATERIALS AND METHODS Chemicals. The chemical names and structures of the tested HO-PBDEs and MeO-PBDEs

92 93

are shown in Table 1 and Figure S1 (Supporting Information), respectively. These chemicals (>98%

94

pure) were synthesized in the Department of Biology and Chemistry of City University of Hong

95

Kong. The methods of synthesis and identification have been described previously29. The

96

5α-dihydrotestosterone (DHT; >99.5% pure) and flutamide (>99% pure) were purchased from Dr.

97

Ehrenstorfer-Schäfers’s laboratory (Augsburg, Germany) and Sigma (St. Louis, MO, USA),

98

respectively. Stock solutions of chemicals were prepared in dimethyl sulfoxide (DMSO; Tedia Co.

99

Ltd, Fairfield, OH, USA) and stored at –20 °C.

100

MDA-kb2 Cell Culture and Reporter Gene Assay. MDA-kb2 cells (catalog number

101

CRL-2713; American Tissue Culture Collection, Manassas, VA, USA) are stably transfected with

102

a luciferase reporter gene, which is driven by an androgen–response element-containing

103

promoter30. The cells were maintained in Leibowitz-15 (L-15) medium (Sigma, St. Louis, MO,

104

USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen Corporation, Carlsbad,

105

CA, USA) in a humidified incubator without additional CO2 at 37 °C 31. Prior to the experiments,

106

the

107

charcoal-dextran-stripped FBS (CDS-FBS, Biological Industries Ltd. Israel) instead of 10% FBS

108

for at least 24 hr. The cells were then seeded at a density of 1×105 cells/mL in 384-well white

109

opaque plate (Corning Inc., Corning, NY, USA) with 80 µL of assay media per well and incubated

110

for 24 hr. The cells were then exposed to seven dilutions (from 1×10-8mol/L to 1×10-5mol/L) of

MDA-kb2

cells

were

maintained

in

L-15

medium


supplemented

with

10%


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111

tested chemicals with or without DHT (1 nM). A blank control and a solvent control were

112

presented in each plate. Various concentrations of DHT (from 1×10-13mol/L to 1×10-6mol/L) were

113

included in each plate for quality control. After 24 hr, the exposure medium was removed from

114

each well and 10 µL of 1 × lysis buffer (Promega, Madison, WI, USA) was added per well for cell

115

lysis. After 10 min, 25 µL of luciferase reagent (Promega, Madison, WI, USA) was added per well.

116

Luciferase activity was measured immediately with Synergy H4 hybrid microplate reader (BioTek

117

Instruments Inc., Winooski, VT, USA).The AR antagonist flutamide was used as a positive control

118

for measuring anti-androgenic activity. Each chemical was assayed independently at least 3 times

119

(3 replicate assays) with a minimum of 3 wells per each replicate assay.

120

Structural Models Preparation. The initial molecular structures of tested chemicals were

121

constructed based on the structures of similar chemicals from NCBI PubChem Compound

122

(http://www.ncbi.nlm.nih.gov/pccompound). The geometries were then optimized by density

123

functional theory at the B3LYP/6-311+G(d,p) level with Gaussian 09 program

124

model of the apo form of AR-LBD (Figure 1A) has been built by homology modeling in

125

SwissModel Workspace (http://swissmodel.expasy.org/workspace/).33-35 The crystal structure of

126

AR-LBD in complex with DHT (PDB entry: 1T7T; http://www.rcsb.org/pdb/) was used as a

127

template for the main body of the AR-LBD (residues 669-882 and residues 892-916). The

128

H11-H12 loop (residues 883-891) was built based on the structure of apo ER-LBD (PDB entry:

129

1A52). The Ramachandran plot (Figure S2, Supporting Information) was generated in the

130

Structural Analysis and Verification Server (http://nihserver.mbi.ucla.edu/SAVES/) to evaluate the

131

quality of the built AR-LBD. Then the tested chemicals were docked into the apo AR-LBD by

132

Surflex-Dock program interfaced with SYBYL 7.3. The details and evaluation of our docking


32

. The structural

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133

process has been described previously.36, 37 The top TotalScore conformation of each ligand was

134

selected as the bioactive conformation. Then the receptor and ligand were merged to be a complex

135

for MD simulation.

136

Molecular Dynamics Simulations. The MD simulations were carried out with the

137

GROMACS 4 package38, 39 on an International Business Machines (IBM) Blade cluster system.

138

Prior to simulation, the CHARMM 27 force field

139

using the GROMACS 4 package and SwissParam ( http://www.swissparam.ch/ )

140

was solvated in a box with TIP3P water molecules 43, keeping the boundary of the box at least 10

141

Å away from any protein atoms. Five chloride ions were subsequently added for charge

142

neutralization. The whole system was then energetically minimized by the steepest-descent

143

method

144

volume for 40 ps with position restraints for ligands. The heated systems at 300K were

145

equilibrated for 200 ps with position restraints for ligands and for 1 ns without restraints at 1 bar

146

and 300K. The MD simulations were then performed in the NPT ensemble with periodic boundary

147

conditions. Electrostatic interactions were calculated using the particle mesh Ewald (PME)

148

algorithm, and Van der Waals interactions were accounted for to a cutoff distance of 10 Å. All

149

simulations were carried out for at least 10 ns using a 2fs time step, and snapshots for analysis

150

were saved every 2 ps.

40, 41

was applied to all structural models by 42

. The model

44

. The minimized systems were then gradually heated from 0 to 300K at a constant

45

151

Data Analysis. The statistical analysis was performed in SPSS 16 (SPSS Inc., Chicago, IL,

152

USA). All results from the reporter gene assay were expressed as the mean ± standard deviations

153

of three independent experiments. One-way analysis of variance (ANOVA) and Duncan’s multiple

154

comparisons test were performed to assess the significance of the differences, and a difference was



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155

considered significant at a p-value≤0.05. The dose–response studies were also subjected to

156

regression analysis using a sigmoidal curve fitting model: Reponse minimum

1

maximum minimum

10 !"#$%&!'"

1#

157

Where ECmedian is the median effective concentration and Hillslope is a slope constant. The results

158

for the AR antagonistic activities of tested chemicals were expressed as 20% relative inhibitory

159

concentration (RIC20, the concentration of the tested chemicals showing 20% inhibition of the

160

activities induced by DHT) based on equation 1.

161

The data from MD simulation was also analysis in GROMACS 4. The root mean square

162

fluctuation (RMSF) of a certain atom was calculated according to the following equation: ; 6

7

1 6 RMSF& , ./01 2t 4 5 01 0/ < 2# T 89 :;

163

Where T is the time over which to be averaged, ri(tj) and ri(0) are the coordinates of particle i at

164

time tj and the initial time, respectively. The RMSF is a measure of the deviation between the

165

position of particle i and some reference position over a period of time. And the root mean square

166

deviation

167

of superimposed proteins. Prior to calculate the RMSD of particles at certain times, the backbones

168

of analyzed AR-LBD and apo AR-LBD were superimposed. Then RMSD was calculated

169

according to the equation 3:

(RMSD)

is

the

measure

B

of

the

average

distance

between

the

atoms

; 6

1 RMSD8 ? .‖01 t 01 0‖6 C 3# N &:;

170

Where N is the total number of analyzed atoms, ri(t) and ri(0) are the coordinates of atom i at time

171

t and initial, respectively. The settling time is defined as time required for the RMSD curve to

172

become stable and it is only expressed as multiples of 0.5 ns.


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173 174

RESULTS AND DISCUSSION

175

Androgenic and Anti-androgenic Activities of HO-PBDE and MeO-PBDE in Reporter

176

Gene Assay. The reliability and sensitivity of the system used in this study were assessed. The

177

dose–response curve for DHT obtained from the reporter gene assay is presented (Figure S3,

178

Supporting Information). The androgenicity of 16 chemicals was examined, and none of them

179

showed any AR agonistic activity (data not shown). However, 6´-HO-BDE-17, 6´-MeO-BDE-17,

180

6-MeO-BDE-47,

181

3-MeO-BDE-100, 2-HO-BDE-123 and 4´-MeO-BDE-49 inhibited the agonistic activity induced

182

by 1.0×10-9 mol/L DHT. The dose responses of the antagonistic activity via the AR for the 16

183

chemicals are shown in Figure 2 and the RIC20 values are shown in Table 1. The antagonistic

184

activities of 4´-HO-BDE-49, 4´-MeO-BDE-49 and 6-MeO-BDE-47 have been detected in

185

previous studies,16,17 and the results were consistent with our results. The activities of other 7

186

chemicals were firstly detected. The chemical of 6´-MeO-BDE-17 (RIC20=0.897 μ mol/L)

187

possessed the highest anti-androgenic potency among the 10 active chemicals. Taking these results,

188

the in vivo effects of these chemicals in human and wildlife are also needed to be further

189

characterized. Moreover, more attention should be given for their environmental/biological effects.

190

The chemical structure of HO-PBDEs and MeO-PBDEs is highly similar. Some of them possess

191

anti-androgenic activity while others not, and the mechanisms behind this activity have not been

192

fully elucidated. It implies that predicting the activity based on the substituent groups or the

193

number of bromine atoms is difficult to achieve, which challenges the risk assessment of

194

HO-/MeO-PBDEs. The cytotoxicity from the MTT assay is shown in the supporting information

4´-HO-BDE-49,

6-MeO-BDE-90,

6-MeO-BDE-85,


6-HO-BDE-90,


195

for quality control.

196

The Importance of Helix 12. For the first time, the stable reposition of H12 is determined as

197

a computational mark for identifying antagonists. The reposition of H12 plays an essential role in

198

the transcriptional activity of NRs. The RMSF values (0-10 ns) of the main-chain atoms of all run

199

systems are shown in Figure 3 and Figure S4 (Supporting Information). A greater RMSF implies

200

that the atoms move farther away from the apo conformation. It is obvious that H12 and the

201

connected loop are the most fluctuating parts of AR LBD. The RMSFs of H1-H11 are lower than 4

202

Å mostly, demonstrating that these parts are stable. Based on this observation, the subsequent

203

analyses of the large MD simulation data focused on H12. The value of RMSD represents the

204

average distance between the atoms of superimposed proteins. A stable RMSD implies that the

205

corresponding atoms become stable, while a fluctuating RMSD implies the fluctuation. The

206

RMSD of the positive control flutamide-H12 (the item of ligand-H12 will be used in this paper to

207

represent the H12 of AR-LBD which occupied by ligand) becomes stable at approximately 2 ns

208

(Figure 4A), while the RMSD of the blank-H12 (H12 of AR-LBD without any ligands) keeps

209

fluctuating (Figure 4B). Keeping consistent with this phenomenon, the H12s of LBDs occupied by

210

10 activated chemicals (6´-HO-BDE-17, 4´-HO-BDE-49, 6-HO-BDE-90, 2-HO-BDE-123,

211

6´-MeO-BDE-17, 6-MeO-BDE-47, 4´-MeO-BDE-49, 6-MeO-BDE-85, 6-MeO-BDE-90 and

212

3-MeO-BDE-100) and 6-HO-BDE-137-H12 are stable before 10 ns (Figure 4A and Figure S5 A,

213

B, Supporting Information). In contrast, when the other metabolites of PBDE (6-HO-BDE-47,

214

6-HO-BDE-85, 3-HO-BDE-100, 2-MeO-BDE-123 and 6-MeO-BDE-137) occupy the LBD, a

215

stable H12 is not achieved, even when the simulation was performed for 15 ns (6-HO-BDE-47 and

216

6-HO-BDE-85 are shown as examples in Figure 4B, the RMSDs of another 3 chemicals are shown


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in Figure S5 C, Supporting Information). The RMSD of 6-HO-BDE-137-H12 stabilizes before 10

218

ns (Figure S5 B, Supporting Information), although no activity was detected. The stable

219

conformations achieved for 7 active ligands occupied AR-LBDs are similar to the conformation of

220

the flutamide occupied AR-LBD (Figure S6 A). For example, the 6´-HO-BDE-17-H12 is near the

221

flutamide-H2 which locates in the surface composed by H3 and H11 (Figure 1C). However,

222

6-MeO-BDE-85-H12, 6-HO-BDE-90-H12, 3-MeO-BDE-100-H12 and 6-HO-BDE-137-H12

223

forward inside, which are different from the flutamide-H12 (Figure 1D and Figure S6 B,

224

Supporting Information). Here, we define the stable conformation of the flutamide occupied LBD

225

and that of 7 analogs as Mode 1. The other stable conformation is defined as Mode 2. The major

226

difference between Mode 1 and 2 is the position of H12, the details are described in Figure 1.

227

The results of the MD simulation and data analysis highlight the reposition of H12 in

228

inducing anti-androgenic activity. It may due to the recruitment of co-repressors. For examples,

229

the conformations of Mode 1 and Mode 2 are suitable for co-repressors such as NcoR and SMRT

230

to recruit to AR-LBD, thus blocking the co-activator recruitment

231

activation is reduced

232

therefore the explanation is uncertain. Another more intuitive explanation relates to the unbinding

233

pathway which is defined as the way ligands get away from AR-LBD. It is well known that the

234

binding of DHT to the LBD is the first and necessary step in the AR-induced transcriptional

235

activation of MDA-kb2. When an exogenous ligand stably occupied the cavity of AR-LBD, the

236

binding efficiency of DHT with AR-LBD was reduced. Thus, the transcriptional activities will not

237

be induced entirely. No study has focused on the unbinding pathway of AR, but the one of ER has

238

been studied 48. An important pathway locates between H11-H12 loop and the N-terminal part of

47

46

. As a result, transcriptional

. However, the antagonistic form of AR-LBD has not been resolved, and



239

the H3. For the Mode 1 conformation of AR-LBD, the H12 folds and struggles the H11-H12 loop

240

to cover the gap between N-terminal region of H3 and C-terminal region of H11 (Figure 1).

241

Another unbinding pathway of ER is located between the H7-H8 loop and the Helix11. For the

242

Mode 2 conformation of AR-LBD in this research, the corresponding position is occupied by the

243

H12 with H11-H12 loop. This positioning may result in the ligands occupying the AR-LBD,

244

causing the activities induced by DHT to be inhibited.

245

A contradiction between the results of the reporter gene assay and the MD simulation was

246

discovered for 6-HO-BDE-137. No activity of this chemical was detected in our assay, but the

247

RMSD became stable at 6ns which implied this chemical possess anti-androgenic activity. This

248

discrepancy may be attributed to the cytotoxicity of this chemical. For the tested chemicals

249

without cytotoxicity, the anti-androgenic activities could be examined explicitly. However, for

250

6-HO-BDE-47, 6-HO-BDE-85 and 6-HO-BDE-137, cytotoxicity was detected at the two highest

251

concentrations. Thus, we could not confirm whether these chemicals possess anti-androgenic

252

activity at these concentrations.

253

Analysis of Dynamic Trajectory. The view of taking ligand and receptor as a dynamic

254

complex is highlighted. The trajectory-analyses of all systems were performed. The trajectories of

255

6´-HO-BDE-17 occupied AR-LBD from 0 to 3.5 ns (settling time) and the 6´-MeO-BDE-17

256

occupied AR-LBD from 0 to 3.5 ns are shown in Figure 5. Their stable conformations are similar,

257

and the H12s are located at almost the same position, but the trajectories of the H12s are

258

dramatically different. The other systems of both Mode 1 and Mode 2 were also analyzed, and the

259

trajectories of the H12s are also not same (data not shown and further details can be obtained from

260

the authors upon request). In addition, H12s of the blank and 6 inactive ligands binding AR-LBD


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261

keep moving which is consistent with the result of RMSD-analysis, in these cases, and never

262

repositioned to the position of Mode 1 H12 or Mode 2 H12 from 0 to 15 ns. The approaches of

263

traditional QSAR and docking are used to understand the ligand-receptor binding mechanism and

264

predict activity based on the molecular features of chemicals, key amino acids of the receptor and

265

some special interactions. Environmental scientists have gained a lot of achievements according to

266

the principle

267

applicable. The trajectories of the H12s that occupy the same final position are dramatically

268

different because the interactions between different ligands and the AR-LBD are different.

269

Meanwhile, the most dynamic amino acids are also different in these complexes, which can also

270

be explained by the immensely different interactions between different ligands and the AR-LBD.

271

This result proves that chemicals can exhibit anti-androgenic activity by different modes of action

272

with the AR-LBD, though the tested chemicals are structurally similar. The key amino acids and

273

key interactions (e.g. Van der Waals interaction or Hydrophobic interaction) can be different for

274

different ligands and this idea has not been fully explored. Some key amino acids of AR-LBD for

275

AR antagonistic effect were proposed in many studies49, 52, 53 by comparative molecular field

276

analysis (CoMFA) or comparative molecular similarity indices analysis (CoMSIA). However, the

277

results from different studies varied widely. These discrepancies are reasonable because the key

278

amino acids of the AR-LBD must be different for different classes of chemicals based on our

279

research. Studies have focused on the hydrogen bonds between the AR-LBD and ligands in hopes

280

of discovering the mechanism of the anti-androgenic effect and predicting this activity

281

example, Bisson et al. emphasized the necessity of the hydrogen bond between ligands and residue

282

Arg752 for AR antagonistic activity 21. However, in this study, the hydrogen bond was not found

49-51

. However, our results suggested that this principle may not be universally


21, 52

. For


283

between Arg752 and all tested chemicals with an anti-androgenic effect. The computed frequency

284

for the formation of the hydrogen bond between some ligands and Leu704 is higher than 80%

285

which can be considered stable (Table S1), while no stable hydrogen bond was found in other

286

cases. Furthermore, no consistency was found between the presence of a stable hydrogen bond and

287

activity. Thus, we propose hydrogen bonding between the AR-LBD and ligands may not be a key

288

factor of anti-androgenic effect, especially for environmental weak agonists and antagonists.

289

Naturally, caution should be taken when elucidating and predicting the anti-androgenic activity of

290

chemicals with these traditional approaches.

291

Relationship between RIC20 and Settling Time. Some attempts have been made to

292

develop the relationship between the RIC20 and allosteric factors. The settling time were

293

calculated based on the analysis of RMSD (Table 1), and it varied from 3.5 ns to 8ns. The

294

relationship between RIC20 and settling time was built via one-parameter linear regression

295

equation (Figure 6). The resulting equation 4 exhibits very good correlation (R2=0.642). RIC6H 1.4027 $ settling time 4.2172 4#

296

The positive coefficient for settling time indicates that increased interval time of H12 from

297

apo conformation to stable conformation leads to a greater RIC20 which implies lower activity.

298

The stable conformation of the AR-LBD and the RMSD of H12 can be utilized to explain the

299

anti-androgenic activity qualitatively. However, it is also important to understand the differences

300

in the RIC20 among the active ligands. The effects on the activity of the AR in cell lines occur on

301

the scale of hours or days for both agonists and antagonists. Even in cell-free systems, the

302

ligand-AR binding process also involves several hours 54. However, the conformational transition

303

in this study is only a nanosecond-scale process. The regression analysis shows a positive


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Page 15 of 33


304

correlation between the RIC20 and settling time, implying that the difference of microscopic

305

conformational transition can lead to difference of activity in a statistical approach. The result

306

indicated that the rapider reposition of H12 exhibited, the faster ligand stabilizing in AR-LBD.

307

Thus, more molecules of DHT are blocked from the LBD and the DHT-induced activity is

308

suppressed more. In fact, the AR signaling involves a cycle and therefore a protein molecule of

309

AR can mediate activity repeatedly

310

should be amplified according to the cycle. Therefore, the settling time of H12 RMSD was

311

proposed to be used to semi-quantitatively predict the potency of AR antagonistic chemicals.

55

. The effect of rapid reposition on anti-androgenic activity

312

Summary and Environmental Significance. The developed methods which aim to detect

313

AR agonists and antagonists are always time-consuming and difficult to conduct30, 54, 56. The kinds

314

of existing chemicals are far more than the ones people can test. These difficulties limit the safety

315

and risk assessment of the anthropogenic chemicals. In silico-aided prediction is a good

316

complementary and alternative approach for detecting anti-/androgenic activity

317

methodology based on MD simulation developed in this study is helpful for promoting in

318

silico-aided prediction. The speed of the GROMACS simulation package is very fast

319

high performance scientific computing techniques are promoted and popularized quickly. Thus,

320

using this package for massive in silico-aided prediction is achievable. A freely accessed

321

web-server will be built for predicting the anti-androgenic effect of HO-PBDEs and MeO-PBDEs

322

(Website: http://hjxy.nju.edu.cn/yuhx/ArticleShow.aspx?ID=90). This computational tool can be

323

used to advance the safety and risk assessment for global researchers.

57

. The

39, 58

, and

324

Having characterized the interaction of metabolites of PBDE with the AR by a reporter gene

325

assay and MD simulation, the HO-/MeOPBDEs were verified as potential environmental EDCs.



326

More studies should be given to the structural family for comprehensive risk assessment.

327

Meanwhile, the stable reposition of H12 in AR-LBD was determined as computational mark for

328

screening AR antagonistic chemicals. The concept of the settling time of RMSD was introduced in

329

this study and characterized as a decisive factor of anti-androgenic potency. Based on these results,

330

we reasoned that knowledge regarding the reposition of H12 and anti-androgenic activity could

331

aid in the development of a computational method to predict binding mode of any analogues of

332

PBDE to AR.

333

Future developments of this method will allow examination of (i) other chemical structural

334

classes, (ii) other members of the NR superfamily, especially the ER, and (iii) even other species,

335

including mouse and zebrafish, for ecological protection. Performing similar and related

336

simulations would be beneficial in understanding the mechanism of endocrine-disruption. As the

337

computational method is cost-effective and independent of the high-purity chemical samples, it

338

will promote more efficient use of risk assessment of new chemicals and emerging pollutants.

339 340

ASSOCIATED CONTENT

341

Supporting Information Available

342

The methods and results of Cytotoxicity test, analysis of hydrogen bonds between ligands and

343

Leu704 (Table S1), molecular structures of 16 chemicals in the current study (Figure S1), the

344

justification of using RIC20, the Ramachandran plot of built apo AR-LBD structure (Figure S2),

345

the relative luciferase activity induced by DHT in reporter gene assay based on MDA-kb2 cell

346

lines (Figure S3), RMSF of the main-chain atom of 15 AR-LBDs (Figure S4), RMSDs of the

347

backbone atoms of H12s which are not presented in manuscript (Figure S5), structure of Mode1


Page 16 of 33

Page 17 of 33


348

and Mode2 of androgen receptor ligand binding domain (AR-LBD) with apo conformation

349

(Figure S6) are included as Supporting Information. This information is available free of charge

350

via the Internet at http://pubs.acs.org.

351 352

AUTHOR INFORMATION

353

*Corresponding Author

354

Wei Shi, PhD: School of the Environment, Nanjing University, Nanjing, 210023, China. Tel.:

355

+86 25 8968 0356, Fax: +86 25 8968 0356, E-mail: [email protected]

356

Prof. Hongxia Yu, PhD: School of the Environment, Nanjing University, Nanjing, 210023, China.

357

Tel.: +86 25 8968 0356, Fax: +86 25 8968 0356, E-mail: [email protected]

358

Notes

359

The authors declare no competing financial interest.

360 361

ACKNOWLEDGEMENT

362

We thank three anonymous reviewers for their constructive suggestions on this paper. This work

363

was supported by National Natural Science Foundation (21397954), Science Foundation in

364

Jiangsu Province (BK20130551&BK2011032), Jiangsu Provincial Environmental Monitoring

365

Research Fund (Grant No. 1212) and Major Science and Technology Program for Water Pollution

366

Control and Treatment of China (2012ZX07101-003). The numerical calculations in this paper

367

have been done on the IBM Blade cluster system in the High Performance Computing Center

368

(HPCC) of Nanjing University. The HO-PBDEs and MeO-PBDEs were kindly provided by

369

Michael H. W. Lam (Department of Biology and Chemistry, City University of Hong Kong).



370

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525

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530

folding. Science 2004, 305, 1605-1609.



531 532 533

Tables Table 1. The name, anti-androgenic activity and settling time of tested chemicals Chemical

RIC20(µmol/L)

Settling time (ns)

6´-HO-BDE-17

1.095

3.5

6-HO-BDE-47

—

—

4´-HO-BDE-49

5.0477

7

6-HO-BDE-85

—

—

6-HO-BDE-90

1.905

5.5

2-HO-BDE-123

4.78

6

3-HO-BDE-100

—

—

6-HO-BDE-137

—

8

6´-MeO-BDE-17

0.897

3.5

6-MeO-BDE-47

1.032

5.5

4´-MeO-BDE-49 6-MeO-BDE-85

8.511 4.522

7 5

6-MeO-BDE-90

4.315

7

2-MeO-BDE-123

—

—

3-MeO-BDE-100

7.079

8

6-MeO-BDE-137

—

—


Page 26 of 33

Page 27 of 33


Figures

534 535 536

Figure 1. Structure of the androgen receptor ligand binding domain (AR-LBD): apo conformation

537

or in complex with ligand. (A) Apo conformation of AR-LBD and H3 (aquamarine), H11 (yellow)

538

and H12 (red) are marked. (B-D) Three conformations are superimposed in every panel, H1-H11s

539

are substantially overlapped 25, but the positions of the H12s vary widely. The H12 of the apo

540

AR-LBD (red), stable flutamide-H12 (blue) and blank-H12 at 10ns (B, slate) are chosen as

541

references to present stable 6´-HO-BDE-17-H12 (C, orange, Mode 1) and stable

542

6-MeO-BDE-85-H12 (D, purple, Mode 2).

543 544

Figure 2. Anti-androgenic effects for tested chemicals and flutamide determined by the MDA-kb2

545

in the concentrations of no cytotoxic effect. The induction activities of HO- and MeO-PBDEs with

546

1.0×10-9 mol /L DHT are represented as fold relative to the maximum induced by DHT. All the

547

values were determined in triplicate. Error bars indicate the standard deviation (SD). * p < 0.05

548

(ANOVA) compared with 1.0×10-9 mol /L DHT alone.

549 550

Figure 3. RMSF of the main-chain atom of 6´-HO-BDE-17 occupied AR-LBD. H12 and the

551

connected loop (red curve) are the most fluctuating parts, and H1-H11 is stable (blue curve).

552 553

Figure 4. RMSD of the backbone atoms of H12s. Five ligand-H12s and blank-H12 are selected as

554

representatives. RMSDs of flutamide-H12, 6´-HO-BDE-17-H12 and 6´-MeO-BDE-17-H12



555

become stable before 10ns (A), but RMSDs of blank-H12, 6-HO-BDE-47-H12 and

556

6-HO-BDE-85-H12 keep fluctuating (B).

557 558

Figure 5. Overlay of snapshots of the conformational dynamics of the AR-LBD occupied by

559

6´-HO-BDE-17 (A) and 6´-MeO-BDE-17 (B). H1-H11s are shown in green, the H12 of apo

560

conformation is shown in red. (A) 6´-HO-BDE-17-H12 is shown in yellow at 1ns and in blue at

561

3.5ns. (B) 6´-MeO-BDE-17-H12 is shown in pink at 1ns and in slate at 3.5ns. The apparent

562

difference between these trajectories is presented.

563 564

Figure 6. Correlations between RIC20 and the settling time of RMSD (H12).

565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588


Page 28 of 33

Page 29 of 33


589 590

Figure 1.

591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606



607

Figure 2

608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637


Page 30 of 33

Page 31 of 33


638 639

Figure 3.

640 641 642 643 644 645

Figure 4

646



647 648

649 650 651 652 653

Figure 5.

Figure 6.

654 655 656 657 658 659 660 661 662


Page 32 of 33

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663 664

Table of Contents (TOC) Art

665


MeO-PBDEs on Androgen Receptor: In Vitro

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