Interactions of fipronil in fish and insects: experimental and molecular

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Agricultural and Environmental Chemistry

Interactions of fipronil in fish and insects: experimental and molecular modeling studies Bo Zhang, lei zhang, lujue he, xiaodong yang, yali shi, shaowei liao, shan yang, Jiagao Cheng, and Tianrui Ren J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00573 • Publication Date (Web): 07 Apr 2018 Downloaded from http://pubs.acs.org on April 9, 2018

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Journal of Agricultural and Food Chemistry

1

Interactions

of

fipronil

within

fish

and

2

experimental and molecular modeling studies

insects:

3 4

Bo Zhang, §,1 Lei Zhang, §,1 Lujue He, §,2 Xiaodong Yang,1 Yali Shi,1 Shaowei

5

Liao,1 Shan Yang,1 Jiagao Cheng,2,* Tianrui Ren1,*

6 7

1. The Key Laboratory of Resource Chemistry of Ministry of Education, The

8

Development Centre of Plant Germplasm Resources, College of Life and

9

Environmental Science, Shanghai Normal University, 100 Guilin Road, Shanghai,

10

200234, P. R. China

11

2. Shanghai Key Laboratory of Chemical Biology, Shanghai Key Laboratory of New

12

Drug design, School of Pharmacy, East China University of Science and Technology,

13

130 Meilong Road, Shanghai, 200237, China

14

§

15

*Corresponding authors:

16

Prof. Tianrui Ren

17

Tel: +86-21-64328850; Fax: +86-21-64328850

18

E-mail: [email protected]

19

Prof. Jiagao Cheng

20

Tel: +86-21-64251348; Fax: +86-21-64252603

21

E-mail: [email protected]

These authors contributed equally to this paper

22

1

ACS Paragon Plus Environment


23

Abstract

24

Fipronil is an efficient phenylpyrazole insecticide that acts on insect

25

gamma-aminobutyric acid (GABA) receptors (GABARs) and has low toxicity to

26

mammals but high toxicity to non-target organisms such as fish. To develop novel

27

efficient low-toxicity insecticides, it is necessary to determine the detailed toxic

28

mechanism at the molecular target level. In this work, methods including affinity

29

chromatography, fluorescent-labeled binding assays and molecular modeling were

30

integrated to explore the binding of fipronil to GABARs in fish (A. nobilis) and

31

insects (M. domestica). Affinity chromatography revealed that fipronil acts on two

32

different subunits of GABARs in fish and M. domestica. Moreover, fluorescence

33

assays revealed that fipronil exhibits similar affinity to the two GABARs. The Kd and

34

Bmax of fipronil binding to the A. nobilis GABAR were 346 ± 6 nmol/L and 40.6 ± 3.5

35

pmol/mg protein, respectively. And the Kd and Bmax of fipronil binding to the GABAR

36

in M. domestica brain were 109 ± 9 nM, and 21.3 ± 2.5 pmol/mg protein, respectively.

37

In addition, similar fipronil binding positions but different binding modes were

38

observed in docking studies with B. rerio var. and M. domestica GABARs. These

39

findings indicated similar interactions of fipronil with fish and insects leading to high

40

toxicity. The different binding features of fipronil between the two species might be

41

helpful for the design and development of highly selective insecticides with low

42

toxicity to fish.

43

Keywords: Fipronil, GABA receptors, affinity chromatography, fluorescence analysis,

44

homology modeling

45 2


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Introduction

47

Fipronil, a broad-spectrum phenylpyrazole insecticide, has been widely used in pest

48

control and veterinary drugs.1,2 The bioactivity of fipronil is ascribed to its ability to

49

target gamma-aminobutyric acid (GABA) receptors (GABARs) and act as a

50

noncompetitive blocker of the GABA-gated chloride channels in the central nervous

51

system.3-5 In addition, fipronil displays greater affinity for insect GABARs than for

52

vertebrate GABARs. Consequently, fipronil has excellent selective toxicity towards

53

insects over mammals.6,7 However, toxicology studies have shown that fipronil

54

displays high toxicity to various non-target aquatic organisms such as fish,8,9 severely

55

restricting its usage. Although the severe toxicity of fipronil to fish has attracted

56

extensive attention, the underlying mechanism at the target level remains unclear.

57

Therefore, it is crucial to elucidate the mechanism of toxicity of fipronil to fish to

58

develop highly selective, safe, and efficient pesticides with low toxicity to fish.

59

Receptor-binding assays are versatile for investigating drug-receptor interactions,

60

and fluorescent probe techniques have emerged as a facile means of investigating

61

interactions between benzodiazepines and GABARs in the mammalian brain.10,11

62

However, fluorescent probe techniques have rarely been used to evaluate the

63

interactions of fipronil with GABARs in fish. Fluorescein is a viable labeling reagent

64

because of its superior fluorescence intensity, high quantum yield, and high stability

65

in alkaline aqueous solutions.12 Fluorescein reacts easily with amino groups and other

66

reactive groups to produce a fluorescent probe for protein labeling.13

67

Here, we report preliminary results on the binding capacity of fipronil to GABARs

68

in the brains of fish and M. domestica based on affinity chromatography, fluorescent 3



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probe analysis and molecular modeling. The interactions between fipronil and the

70

GABARs from both species were systematically investigated. Exploring the different

71

binding features of fipronil between these two species may aid the development of

72

highly selective insecticides with low toxicity to fish.

73 74

Materials and methods

75

Chemicals

76

Pesticide

analytical

standards

fipronil,

CL-6B,

bromide

(TBAB),

4-dimethylaminopyridine

78

N,N-dimethyl formamide (DMF), bromoacetyl bromide, 1,4-butanediol diglycidyl

79

ether and other chemicals were purchased from Aladdin. Double-distilled water was

80

used in the experiments. All chemicals were of analytical grade and were used without

81

further purification. A. nobilis brain was obtained from fresh fish markets in Shanghai,

82

China. Brain tissue was kept at -70 °C until use.

83

Preparation of the media for fipronil affinity chromatography

84

Synthesis of the fipronil affinity ligand

86

tetrabutylammonium

Sepharose

77

85

(DMAP),

of

The fipronil affinity ligand (compound 3) was prepared according to the literature.14 The synthesis route for compound 3 is presented in Scheme 1.

87

Synthesis of Compound 1. A solution of fipronil (4.40 g, 10 mmol) in CH2Cl2

88

(30 mL) was cooled to 0 °C under a nitrogen atmosphere and treated with a solution

89

of DMAP (1.2 g) in trimethylamine (4 mL). The mixture was stirred for 5 min, and

90

bromoacetyl bromide (4 mL, 46 mmol) was added dropwise. The resulting solution

4


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91

was stirred for 8 h at room temperature. The reaction mixture was quenched by

92

adding ice water and extracted with CH2Cl2 (20 mL×3). The combined organic layers

93

were washed with brine, dried over MgSO4, filtered, and evaporated in vacuo. The

94

residues were purified by column chromatography on silica gel (petroleum

95

benzine:acetidine = 8:1 (v/v)) to obtain product 1 as a yellow solid in 84% yield. m.p.

96

167-169 °C; 1H NMR (CDCl3, 600 MHz), δ 9.22 (s, 1H, NH), 7.73-7.80 (s, 2H,

97

Ar-H), 4.11-4.12 (s, 2H, CH2).

98

Synthesis of Compound 2. Compound 1 (2.32 g of 4 mmol), phthalimide

99

potassium salt (7.4 g, 40 mmol), TBAB (0.8 g), and DMF (40 mL) were mixed by

100

vigorous magnetic stirring at 80 °C. The reaction mixture was maintained at 80 °C for

101

6 h. The resulting reaction mixture was poured into ice water and extracted with

102

CH2Cl2 (3 × 20 mL). The organic layer was separated, washed with brine, and dried

103

over MgSO4. The solvent was removed by distillation in a vacuum. The obtained

104

residue (compound 2) was used without further purification.

105

Synthesis of Compound 3 (affinity ligand): Compound 2 (6.23 g, 10 mmol)

106

was dissolved in 30 mL of ethanol, and 85% hydrazine hydrate (1.18 g, 20 mmol) was

107

added dropwise. The solution was stirred for 5 h at 70 °C. The reaction mixture was

108

then quenched by adding ice water and extracted with diethyl ether (3 × 30 mL). The

109

organic layer was washed with brine, dried over MgSO4, filtered, and evaporated in

110

vacuo. The resulting residue was purified by silica gel column chromatography to

111

obtain compound 3 (affinity ligand) as a yellow solid in 40% yield. m.p. 197-199 °C;

112

1

H NMR (600 MHz, CDCl3) δ: 8.41 (1H, s, NH), 7.72-7.70 (2H, d, Ar-H/H0), 3.10

5



113

(2H, s, CH2), 2.81 (2H, s, NH2);

114

Synthesis of fipronil affinity gel. The synthesis of the matrix used in fipronil

115

affinity chromatography is shown in Scheme 2. Epoxy-activated Sepharose CL-6B

116

(30 mL) was filtered and washed with distilled water. The washed gel was

117

resuspended in 40 mL of sodium carbonate buffer (pH 9.0, 0.1 mol/L), and 10 mL of

118

DMSO containing 0.2 g of the fipronil affinity ligand was added. The mixture was

119

then incubated for 12 h at 37 °C. The gel coupled with fipronil was treated with 1

120

mol/L 2-ethanolamine (pH 8.0) for 4 h at 37 °C and then filtered. After successive

121

washings with 0.1 mol/L (pH 4.0) acetic acid-sodium acetate buffer containing 0.5

122

mol/L NaCl and 0.1 mol/L (pH 8.5) boric acid-sodium borate buffer containing 0.5

123

mol/L NaCl, the resulting gel was finally washed with distilled water, filtered, and

124

stored in 20% ethanol at 4 °C.

125

Procedures for affinity purification. The solubilized receptor preparations from

126

the brains of A. nobilis and M. domestica were obtained according to a previously

127

described method.15 The column (1.0 cm×10 cm) packed with fipronil affinity gel was

128

washed with 100 mL of pre-equilibrating buffer (pH 7.5, containing 10 mmol/L

129

K3PO4, 2 mmol/L magnesium acetate, 50 mmol/L KCl, 11% (w/v), 1 mmol/L EGTA

130

and 0.3% (w/v) Triton X-100). Then, 30 mL of the solubilized receptor preparation

131

was applied and incubated for 15 min. Hybrid proteins were eluted with buffer B (pH

132

7.5, containing 0.02 mmol/L K3PO4, 11% (w/v) sucrose, 2 mmol/L magnesium

133

acetate, and 0.3% (w/v) Triton X-100) at a flow rate of 40 mL/h. The receptor protein

134

was bio-specifically eluted with buffer solution C (pH 7.5, containing 0.01 mmol/L

6


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K3PO4, 10 mmol/L flurazepam, 11% (w/v) sucrose, 2 mmol/L magnesium acetate, and

136

0.3% (w/v) Triton X-100) at a flow rate of 20 mL/h.

137

Binding assay for fluorescent probe FF

138

Preparation of fluorescent probe FF. The fluorescent probe FF was prepared as

139

depicted in Scheme 3. A mixture of fipronil (0.36 g, 0.8 mmol), FITC (0.16 g, 0.40

140

mmol), and a catalytic amount of TEA and DMF (10 mL) was incubated with stirring

141

in the dark at 40 °C for 12 h. The organic solvent was evaporated in vacuo. The

142

residues were purified by column chromatography on silica gel (ammonia: methanol:

143

chloroform = 2:33:65). Finally, 0.16 g of FF was obtained as an orange solid in 68.2%

144

yield. 1H NMR (600 MHz, CD3OD) δ: 6.63 (m, 1H), 6.66 (d, 3H), 6.86 (d, 1H),

145

6.90-7.04 (m, 1H), 7.60 (d, 1H), 7.95 (d, 2H), 8.00 (t, 1H), 8.08 (s, 1H). MS-ESI, m/z:

146

calcd. for [FF+H]+ 826.53, found 826.06.

147

Separation of the receptor membrane preparations. Brains of A. nobilis and M.

148

domestica were obtained according to the methods of Janssen et al.,16 and then

149

homogenized in a glass homogenizer in Tris-HCl buffer (pH = 7.5, 50 mmol/L). All

150

operations were performed at 0-4 °C. The homogenates were centrifuged at 1000 g for

151

15 min. The supernatant was then centrifuged at 1.5 × 105 g for 40 min. The pellets

152

were resuspended in the buffer solution and centrifuged at 1.5 × 105 g for 60 min.

153

Three replicates were used for each series of experiments. The obtained samples were

154

stored at -80 °C until use. The protein concentration was determined by the Bradford

155

method.

156

Binding assay of fluorescent probe FF to the receptor membrane preparation.

7



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157

Receptor-binding assays were performed as follows. For the saturation experiments,

158

50 µL of FF solution (2.5-250 nM final concentration) was added to 930 µL of the

159

receptor membrane preparations. The protein concentrations of the brains of A. nobilis

160

and M. domestica were 605.20 and 318.68 µg, respectively. For the determination of

161

total or nonspecific binding, 20 µL of Tris-HCl buffer solution (pH 7.4) or 20 µL of

162

1.0 mM fipronil solution, respectively, was added to each EP tube. To this mixture,

163

930 µL of receptor membrane preparation was added to obtain a total volume of 1

164

mL. The mixture was incubated in each EP tube for 1 h at 4 °C. After centrifugation at

165

22000 g for 15 min (4 °C), the precipitate was rinsed twice with 1 mL of Tris-HCl

166

buffer (pH=7.5, 50 nmol/L), and the filtrate was collected (S1). The precipitate was

167

resuspended in 1 mL of Tris-HCl buffer (P1) and then centrifuged at 20000 g for 10

168

min. The obtained precipitate was added into 1 mL of 50% methanol aqueous solution

169

(v/v) to dissociate the bound FF, and then centrifuged. The pellet was discarded and

170

the supernatant (S2) was collected. The fluorescence intensities of S1, P1, and S2

171

were determined by fluorescence spectroscopy at a fixed excitation wavelength of 490

172

nm. The emission spectra were recorded at 490-600 nm. The results are the means ±

173

standard deviations for three experiments each with three replicates. Specific binding

174

for FF was determined as the difference between total binding and non-specific

175

binding.

176

Homology modeling. The target sequences of B. rerio var. α1β2γ2 subunits

177

(UniProt ID AAI24698, AAI15079 and XP_687331) and M. domestica RDL (UniProt

178

ID

Q75NA5)

were

retrieved

from

8


Swiss-Prot/TeEMbL

Page 9 of 30


179

(https://www.ncbi.nlm.nih.gov/guide/). Additionally, the crystal structure of the

180

glutamate-gated chloride channel (PDB ID 3RHW) in the open state was chosen as a

181

template to construct the three-dimensional structures of the two GABARs.17

182

The amino acids in the intracellular region of the target sequence were removed

183

during homologous modeling as the intracellular loop region between TM3 and TM4

184

of the ligand-gated ion channel has not been resolved. A sequence alignment program

185

was used to compare the amino acid sequences of the α1, β2 and γ2 subunits of B.

186

rerio var. and the M. domestica RDL subunit with the template sequence. The

187

sequence identities of 3RHW were 35%, 37.8% and 32.6% with the B. rerio var. α1,

188

β2 and γ2 subunits, respectively, and 40.9% with M. domestica RDL. Based on the

189

alignment results (Supporting Information Figure S1-S2), the three-dimensional

190

models were built using the Discovery Studio 2.5 software package. Then, the quality

191

of the established 3D structures was assessed by the PROCHECK18 and the

192

Profile-3D19 approaches.

193

Molecular docking. Ligand docking was performed using the Glide program

194

integrated in Maestro 10.2 with default settings, similar to the procedure used in our

195

previous study.20 The binding site was set around the centroid of the -2’ and 9’

196

residues from five chains with a size of 20 Å. The ligand placed in the binding site for

197

a multi-conformational search, and 100 conformations were output and ranked by

198

GlideScore. The superior pose with a reasonable binding orientation was selected for

199

further analysis.

200

Results and discussion

9



201

Affinity analysis technique. Affinity chromatography is an effective means of

202

investigating the specific interactions of drugs with related acceptors. The affinity

203

ligand is the most important molecular recognition moiety. Here, fipronil, a key

204

pesticide targeting GABARs, served as a high-affinity ligand to fish GABARs from

205

A. nobilis and M. domestica. As shown in Figure 1, two major protein bands were

206

obtained from the brains of A. nobilis and M. domestica using the fipronil affinity

207

column (Scheme 2). Notably, the molecular weights of the bands from both species

208

showed considerable difference. The molecular weights of the two bands obtained

209

from A. nobilis brains were approximately 44 and 55 kD (Figure 1A), of which the 55

210

kD band has been reported generally present in the GABARs of teleostean.21

211

However, two major bands approximately 44 and 50 kD were identified from M.

212

domestica brains (Figure 1B). The band with lower molecular mass at 44 kD from

213

two species was perhaps the proteolytic products of the GABARs.21,22 These results

214

suggested differences in the interactions of fipronil between fish and insects.

215

Binding assay analysis. According to the FTIR spectroscopic data (Supporting

216

Information Figure S3), the fluorescent probe FF was successfully synthesized by

217

reacting fipronil with FITC. Its fluorescence properties (Supporting Information

218

Figure S4) in aqueous solution were similar to those of FITC, with an excitation

219

maximum at 490 nm and an emission maximum at approximately 516 nm. We also

220

determined that the fluorescence intensities of FF were strongly influenced by the

221

solvent system. Moreover, the fluorescence intensity of FF in the solution containing

222

the acceptor protein was significantly higher than that in Tris-HCl buffer and 50%

10


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223

methanol solution due to the background fluorescence of the receptor protein

224

(Supporting Information Figure S5).

225

The binding of FF to GABARs in the brains of A. nobilis and M. domestica was

226

assessed by performing saturation experiments. The results (Figure 2A) showed that

227

the binding of FF to the receptor gradually saturated with increasing FF concentration

228

(0-300 nmol/L). The dissociation constant (Kd) and maximum binding capacity (Bmax)

229

were further obtained from Scatchard analysis of the ligand-receptor interactions

230

(Figure 2B). The obtained Kd and [RT] values are shown in Table 1. It has been

231

reported23,24 that fluorescent ligands bound to GABARs can be dissociated with

232

methanol aqueous solution or acetic acid solution. The Bmax and Kd values were

233

similar to those of the radiolabel, suggesting that dissociation of the fluorescent

234

ligands in acetic acid buffer or methanol aqueous solution was feasible. However, due

235

to the instability of FITC in acetic acid solution, the binding of FF was dissociated

236

using methanol aqueous solution.

237

The maximum Kd value can be obtained by the determination of the free ligand S1,

238

which was calculated to be 502 ± 8 nM. The Kd value obtained from the dissociation

239

of S2 in methanol aqueous solution (50:50, v / v) was minimal, with a value of 346 ±

240

6 nM. In the ligand-receptor binding, the amount of free ligand (S1) was much higher

241

than that of bound ligand (S2). In addition, the background fluorescence of the

242

receptor membrane preparation (P1) greatly influenced the determination of the exact

243

fluorescence intensity of FF. Therefore, the determination of the Kd value of FF

244

dissociation from the receptors was feasible, consistent with literature results.25,26

11



245

We performed saturation experiments for the binding of FF with GABARs from the

246

brains of A. nobilis. Then, the bound FF amount (S2) could be precisely detected from

247

the dissociation study, as compared with the values obtained from the S1, P1. Thus

248

only the S2’ curve was determined in M. domestica system.

249

In addition, the obtained Kd value was far less than that of fipronil binding to

250

mammalian GABARs (Kd = 16 µM in the receptor stimulation state, Kd = 26 µM in

251

the receptor closed state)23 but similar to that of fipronil binding to insect GABARs

252

(Kd = 179 nM in the receptor stimulation state, Kd = 98 nM in the receptor closed

253

state).26 Notably, the Bmax and Kd values of FF binding to the M. domestica GABARs

254

were 21.3 ± 2.5 pmol/mg protein and 109 ± 9 nM, respectively, in good agreement

255

with previous reports from Abalis27 and Rosario28. In the radioisotopic labeling assay,

256

the Kd values of fipronil binding to the GABARs of M. domestica were 24.3 nM and

257

23 nM, respectively.27,28 Therefore, the superior affinity of fipronil for GABARs in M.

258

domestica may underlie the high fipronil toxicity in insects. Moreover, the above

259

results showed that fipronil exhibited high affinity for fish GABARs, which may be

260

responsible for the high toxicity of fipronil to fish. The saturability experimental

261

analysis revealed that fipronil exhibits similar interaction trends to the two GABARs,

262

while fipronil displayed slight different binding potencies with GABARs from fish

263

versus housefly, according to the Kd and Bmax values. It inspired us to explore the

264

detailed binding features of fipronil with the GABARs from different species, to give

265

some clues for future low toxicity insecticide design. Accordingly, further molecular

266

modelling studies were performed.

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Homology models of GABARs.

268

Homology modeling studies were performed to construct the GABAR models. The

269

α1β2γ2 subtype (the major subtype in vertebrates)29 GABAR in zebra fish (B. rerio

270

var) was selected as the representative GABAR in fish and the M. domestica

271

RDL-GABAR was chosen as the typical GABAR in insects. The modeled structures

272

of the GABARs and their quality verification results are displayed in Supporting

273

Information Figure S6-S8.

274

To enable a clear comparison between the two species, the residues in the TM2

275

helices were renumbered as depicted in Figure 3. In the fipronil binding area, the

276

amino acids of the 2’, 6’ and 9’ positions are oriented toward the channel pore and

277

have been reported to play important roles in the binding of fipronil.20,30,31 The

278

residues in the 2' position varied. In the α1, β2 and γ2 subunits of B. rerio var., the

279

amino acids at the 2'-position are Val, Ala and Ser, respectively, whereas in the M.

280

domestica RDL subunit, Ala is in this position. The residues between positions 6' and

281

9' are highly conserved in all subunits of the two GABAR models.

282

Docking Results.

283

Docking studies were performed to investigate the detailed interactions between

284

fipronil and GABARs of fish and insects. The docking results are depicted in Figure 4.

285

The final docking pose was obtained by considering the GlideScore values and

286

analyzing the binding modes. Similar fipronil binding poses were observed between B.

287

rerio var. α1β2γ2 subtype (Figure 4A-B) and M. domestica RDL GABARs (Figure

288

4C-D). The fipronil was surrounded by five TM2 helices, and the trifluoromethyl

13



289

groups were both oriented toward the intracellular domain, consistent with previous

290

findings using zebrafish β3 and fruit fly RDL homopentamers of GABAR models.20

291

The residue 6’Thr has been reported to be very important for the binding of fipronil

292

to GABARs.20,30 In the B. rerio var. α1β2γ2 system, the N−H···O hydrogen bond

293

between the amino group of fipronil and the side chain of 6’Thr is strong, with an

294

H···O distance of 2.1 Å (Figure 4B), whereas in the M. domestica RDL system, the

295

corresponding hydrogen bond is weak, with an H···O distance of 2.8 Å (Figure 4D).

296

Thus, the interaction between 6’Thr and fipronil appears to be stronger in B. rerio var.

297

than in M. domestica. However, in the M. domestica RDL system, a new backbone

298

Cα-H···N hydrogen bond was observed between the 6’Thr of another chain and the

299

nitrile group of fipronil, with an H···N distance of 2.6 Å (Figure 4D and Supporting

300

Information Figure S9). Although the binding poses of fipronil with the different

301

GABARs were similar, the identification of diverse binding features from docking

302

studies might be helpful for designing new phenylpyrazole insecticides with low fish

303

toxicity.

304

In conclusion, three research methods, affinity chromatography, fluorescent-labeled

305

binding assays and molecular modeling, were used to explore the similarities and

306

differences in the interactions of fipronil with GABARs in fish and insects. Affinity

307

chromatography showed that fipronil acts on two different subunits of the GABARs

308

in fish and insects. The fluorescence assay revealed that fipronil exhibited similar

309

affinity to GABARs in fish and M. domestica. The Kd and Bmax of fipronil binding to

310

the A. nobilis GABAR were 346 ± 6 nmol/L and 40.6 ± 3.5 pmol/mg protein,

14


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311

respectively. By contrast, the Kd and Bmax of fipronil binding to the GABAR in M.

312

domestica brains were 109 ± 9 nM, and 21.3 ± 2.5 pmol/mg protein, respectively. In

313

addition, the molecular modeling study revealed similar fipronil binding poses but

314

different binding modes in B. rerio var. and M. domestica GABARs. The similarities

315

of the interactions of fipronil with GABARs in fish and insects may contribute to the

316

serious toxicities of fipronil to fish and insects, whereas the diverse binding features

317

might be beneficial for the design of new phenylpyrazole insecticides with low fish

318

toxicity.

319 320 321

Funding We thank the National Natural Science Foundation of China (21642003, 21572059),

322

the

Innovation

Program

of

Shanghai

Municipal

Education

Commission

323

(201701070002E00037), and the Shanghai Normal University scientific research

324

project (SK201703).

325 326

References

327

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benzodiazepines using coumarin-labeled desethylflumazenil as ligand. Anal.

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development of small ligands for receptor binding studies using fluorescence

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correlation spectroscopy. J. Med. Chem. 2004, 47, 3600-3605.

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applications of fluorescent probes for zinc, ZnAFig. S. J. Am. Chem. Soc. 2002,

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mobility and insecticidal activity. Pestic. Biochem. Phys. 2009, 95, 126-130.

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(15) Sigel, E.; Mamalaki, C.; Eric, A. Isolation of a GABA receptor from bovine

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brain using a benzodiazepine affinity column. FEBS Lett. 1982, 147, 45-48.

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a program to check the stereochemical quality of protein structures. J. Appl.

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Crystallogr. 1993, 26, 283-291.

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insights from homology modeling, docking, and molecular dynamics simulation

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comparison of the photoaffinity-labeled benzodiazepine receptor subunits. J.

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(22) Deng, L.; Nielsen, M.; Olsen, R. W. Pharmacological and biochemical properties

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of the γ-aminobutyric acid-benzodiazepine receptor protein from codfish brain. J.

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Ganglion Neurons. J. Pharmacol. Exp. Ther. 2001, 296, 914-921.

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Biochem. Phys. 1983, 20, 39-48.

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(28) Rosario, P.; Barat, A.; Ramirez, G. Characterization of binding sites for [3H]

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Receptor Subtype Involvement in Addictive Behaviour. Genes Brain Behav.

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(31) Cheng, J.; Ju, X. L.; Chen, X. Y.; Liu, G. Y. Homology modeling of human

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α1β2γ2 and house fly β3 GABA receptor channels and Surflex-docking of

19



420

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421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 20


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443

Figure and Scheme Captions

444

Figure 1. Purification of receptor proteins from A) A. nobilis and B) M. domestica by

445

fipronil affinity chromatography.

446

Figure 2. Saturability A) and Scatchard analyses B) of FF binding to GABARs. S1,

447

S2 and P, A. nobilis GABAR; S2’, M. domestica GABAR.

448

Figure 3. Amino acids of the TM2 region in the B. rerio var. α1β2γ2 and M.

449

domestica RDL GABARs

450

Figure 4. 3D model of fipronil binding with B. rerio var. (A, B) and M. domestica

451

RDL (C, D) GABARs. (A, C) Docking pose of fipronil in the binding site of GABAR.

452

(B, D) The interactions of fipronil with key residues (ribbon: TM2 region; sticks:

453

fipronil and residues 6’Thr). For clarity, one β2 subunit of the B. rerio var. model and

454

one RDL subunit of the M. domestica model are not displayed.

455

Scheme 1 Synthesis of the fipronil affinity ligand.

456

Scheme 2 Schematic illustration of the preparation of the fipronil affinity matrix.

457

Scheme 3 Synthetic scheme for the preparation of the fluorescent probe FF.

458 459 460

Table Captions

461

Table 1 Equilibrium binding analysis of FF binding to GABARs

462 463 464

21



465

Figures

466

467 468

Figure 1. Purification of receptor proteins from A) A. nobilis and B) M. domestica by

469

fipronil affinity chromatography.

470 471 472 473 474 475 476 477 478 479 480 481 22


Page 22 of 30

Page 23 of 30


482

483

B 0.20 S1 S2 P

15 S2'

10

5

0 0

50

100

150

200

250

FFBound/Free(pmol/mg protein)

[FF]bound (pmol/mg protein)

A 20

0.15

0.10

0.05

S1 S2 P

S2'

0.00 0

5

10

15

20

25

30

FFBound(pmol/mg protein)

FF (nm)

484

Figure 2. Saturability A) and Scatchard analyses B) of FF binding to GABARs. S1,

485

S2 and P, A. nobilis GABAR; S2’, M. domestica GABAR.

486 487 488 489 490 491 492 493 494 495 496 497 498 499 23



500 501

Figure 3. Amino acids of the TM2 region in the B. rerio var. α1β2γ2 and M.

502

domestica RDL GABARs.

503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519

24


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520

521 522

Figure 4. 3D model of fipronil binding with B. rerio var. (A, B) and M. domestica

523

RDL (C, D) GABARs. (A, C) Docking pose of fipronil in the binding site of GABAR.

524

(B, D) The interactions of fipronil with key residues (ribbon: TM2 region; sticks:

525

fipronil and residues 6’Thr). For clarity, one β2 subunit of the B. rerio var. model and

526

one RDL subunit of the M. domestica model are not displayed.

527 528 529 530 531 532 25



533

Page 26 of 30

Schemes

534 CF3

CF3 O

Cl N NC

535

N

Cl Br NH2 S CF 3 O

Br 1

CF3

O KN

Cl N NC

N

Cl O Br NH

Cl

Cl O

O N

2 S CF 3 O

NC

Compound 1

CF3

N

N H S CF 3 O

O Cl N 3 O

Compound 2

536 537

Scheme 1 Synthesis of the fipronil affinity ligand.

538 539 540 541 542 543 544 545 546 547 548 549 550 551 552

26


N NC

N

Cl O NH2 NH S CF 3 O

Compound 3

Page 27 of 30


CF3 Cl N NC

553 554

N

CF3 Cl O NH2 NH

OH

+

O

O

O

O

OHSepharose

Cl N

S CF 3 O

NC

N

OH Cl O NH NH O

OH O

O

Sepharose

S CF 3 O

Scheme 2 Schematic illustration of the preparation of the fipronil affinity matrix.

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 27



574 575

Scheme 3 Synthetic scheme for the preparation of the fluorescent probe FF.

576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

28


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594 595

Tables Table 1 Equilibrium binding analysis of FF binding to GABARs Components

Kd (nmol/L)

Bmax (pmol/mg protein)

FF(S1)

502±8

56.2±5.0

FF(P1)

377±7

39.3±3.7

FF(S2)

346±6

40.6±3.5

FF(S2’)

109±9

21.3±2.5

596 597 598 599 600 601 602 603 604 605 606 607 608 609 610

29



611

TOC Graphic

612 613 614

30


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Interactions of fipronil in fish and insects: experimental and molecular

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