Discovery of TAS2R14 Agonists from Platycodon grandiflorum Using

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Bioactive Constituents, Metabolites, and Functions

Discovery of TAS2R14 agonists from Platycodon grandiflorum using virtual screening and affinity-screening based on a novel TAS2R14functionalized HEMT sensor combined with UPLC-MS analysis Zhixin Wang, Yuxin Zhang, Yanling Zeng, Xi Li, Zhao Chen, Jiaming Luo, Yang Zhang, Yanling Zhang, and Yanjiang Qiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04455 • Publication Date (Web): 27 Sep 2018 Downloaded from http://pubs.acs.org on September 30, 2018

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Journal of Agricultural and Food Chemistry

1

Discovery of TAS2R14 agonists from Platycodon grandiflorum using

2

virtual

3

TAS2R14-functionalized HEMT sensor combined with UPLC-MS analysis

screening

and

affinity-screening

based

on

a

novel

4 5

Zhi-Xin Wang,† Yu-Xin Zhang,† Yan-Ling Zeng,† Xi Li,† Zhao Chen,† Jia-Ming Luo,‡

6

Yang Zhang,‡ Yan-Ling Zhang,∗,† and Yan-Jiang Qiao∗,†

7 8

†

9

Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing

Key Laboratory of TCM Information Engineering of State Administration of

10

102488, P. R. China

11

‡

12

Beijing 100083, P. R. China

Key Laboratory of Semiconductor Materials Science, Chinese Academy of Sciences,

13

Corresponding author: Prof. Yan-Jiang Qiao, School of Chinese Materia Medica,

14

∗

15

Beijing University of Chinese Medicine, Yangguang South Street, Fangshan District,

16

Beijing 102488, P. R. China. Phone: 086-10-84738661. E-mail: [email protected]

17

∗

18

Medica, Beijing University of Chinese Medicine, Yangguang South Street, Fangshan

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District, Beijing 102488, P. R. China. Phone: 086-10-84738662. E-mail:

20

[email protected]

Additional corresponding author: Prof. Yan-Ling Zhang, School of Chinese Materia

21

1

ACS Paragon Plus Environment


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ABSTRACT: TAS2R14 is of great potential as a therapeutic target against asthma,

23

and discovery of TAS2R14 agonists can be much valuable for treating this disease.

24

Herein, we developed a strategy using virtual screening and affinity-screening based

25

on a fabricated biosensor combined with UPLC-MS analysis to screen TAS2R14

26

agonists from Platycodon grandiflorum. By ligand-based virtual screening, 16 best-fit

27

candidates were yielded. A novel TAS2R14-functionalized HEMT sensor was applied

28

to detect and fish out the potential TAS2R14 agonists from P. grandiflorum extracts.

29

Those components captured by the immobilized TAS2R14 were eluted and

30

characterized on UPLC-QTOF MS. As results, six potential TAS2R14 agonists were

31

screened out and identified. Among them, platycodin L was confirmed to be a special

32

agonist of TAS2R14 for the first time and had an EC50 of 15.03 ± 1.15 µM via

33

intracellular calcium mobilization assay (n=6). The results indicated that the proposed

34

strategy was efficient to discover TAS2R14 agonists from herb directly.

35 36

KEYWORDS: taste type 2 receptor 14 (TAS2R14), agonists, Platycodon grandiflorum,

37

virtual screening, high electron mobility transistor (HEMT)

38

2


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■ INTRODUCTION

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The bitter taste perception of human is mediated via taste type Ⅱ receptors

41

(TAS2Rs)1. This kind of special G protein-coupled receptor (GPCR) was firstly

42

discovered in gustatory cells, afterwards in extraoral system, where they play various

43

physiological functions2. Especially, the expression and latent functions of TAS2Rs in

44

respiratory tract have drawn broad concerns3-6. Previous investigations have proved

45

that TAS2R14, the first identified one among the 25 subtypes of TAS2Rs, owns an

46

exceptionally wide agonist spectrum7-9 and the highest expression level in human

47

bronchi10. Many TAS2R14 agonists, such as caffeine, quinine and diphenidol have

48

remarkable effects on relaxation of airway smooth muscle, and they were thought to

49

be promising drugs for asthma treatment clinically5,11. Therefore, TAS2R14 is of great

50

potential as a therapeutic target against asthma, and discovery of TAS2R14 agonists

51

can be much valuable for treating this disease.

52

Traditional Chinese medicine (TCM) have been served as important source for

53

discovery of therapeutic agents over the past few decades12. Platycodon grandiflorum

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(Jacq.) A. DC., a kind of drug-food homologous plant, is widely distributed in China,

55

Korea and Japan. The dried root of P. grandiflorum was designated as “bitter taste”

56

medicine according to the herbal property theory13, and has been used to treat

57

respiratory disease such as asthma14, airway inflammation15-16 and pulmonary

58

tuberculosis13, so some chemical components in this herb are likely to be the

59

TAS2R14 agonists with potential clinical effects. High-throughput screening (HTS) is

60

one of the dominant techniques for screening active ingredients from complex herb, 3



61

but its applications in discovering the agonists of TAS2Rs are rare. Hu et al. fished out

62

three TAS2R14 agonists from hundreds of natural compounds using HTS model

63

based on TAS2R14 over-expressed HEK293 cells17. However, this method will be

64

effective only if a large bank of pure compounds are available, namely, HTS entails a

65

huge separation workload and a low efficiency when using TCM as source. Hence, it

66

is quite essential but challenging to develop an efficient and fast screening method for

67

identifying agonists of TAS2Rs from TCM directly.

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As a semiconductor device, AlGaAs/InGaAs-based high electron mobility

69

transistor (HEMT) of heterostructure shows characteristic electronic properties

70

including piezoelectric and spontaneous polarization, which bring about high density

71

and migration rate of two-dimensional electron gas (2DEG) in its heterogenous

72

interface. Because that interface is very close to the surface of device, any variance of

73

surface conditions, such as binding outside molecules to the gate area will

74

significantly change the 2DEG density, which in turn alters the size of interpolar

75

current. Therefore, the HEMT can be taken as a powerful tool with ultra-high

76

sensitivity to detect bound substances18. Moreover, profiting from the advantages of

77

column packed with sub-2 µm particle, ultra-high liquid chromatography (UPLC)

78

enables efficient separation and saves analysis time19. And high-resolution mass

79

spectrometry (HRMS) owns excellent qualitative ability, the combination of UPLC

80

with HRMS has become an extremely important technology in the field of natural

81

product analysis more and more frequently20-21. In this study, an efficient strategy

82

based on virtual screening and affinity-screening by a novel TAS2R14-functionalized 4


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HEMT sensor combined with UPLC-MS analysis was proposed to fish out TAS2R14

84

agonists from P. grandiflorum. By ligand-based virtual screening, 16 best-fit

85

candidates were firstly yielded. Then TAS2R14 in membrane protein extracted from

86

recombinational TAS2R14 over-expressed HEK293 cells was immobilized on

87

AlGaAs/InGaAs HEMT sensor to detect and capture the potential TAS2R14 agonists

88

from P. grandiflorum extracts, followed by the two-step elution to release the bound

89

agonists. The released components were collected and applied to UPLC-QTOF MS

90

analysis for identification. As results, six pentacyclic triterpenoid saponins including

91

platycoside D, platycoside G2, platyconic acid A, platycodin D, platycodin L and

92

platycodin K were identified and considered as the potential TAS2R14 agonists,

93

which were also the hits in virtual screening. Among them, platycodin L was

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confirmed to be a special agonist of TAS2R14 for the first time and had an EC50 of

95

15.03 ± 1.15 µM via intracellular calcium mobilization assay. This strategy could

96

facilitate the research and development of effective anti-asthmatic agents and provide

97

a useful reference for screening agonist or inhibitor class of drugs from TCM directly.

98 99

■ MATERIALS AND METHODS

100

Chemicals and Materials

Dulbecco’s modified eagle medium (DMEM), fetal

101

bovine serums (FBS), bull serum albumin (BSA), tris buffered saline with Tween-20

102

(TBST), phosphate buffer solution (PBS, 100 mM, pH 7.4), Hank’s balanced salt

103

solution (HBSS), penicillin-streptomycin mixed solution (100×), hygromycin B,

104

geneticin (G418) and Mem-PER™ plus membrane protein extraction kit (89842) 5



105

were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bicinchoninic

106

acid protein assay (BCA) kit was from Biomiga (San Diego, CA, USA). Rabbit

107

polyclonal anti-TAS2R14 antibody was from Abcam (Cambridge, UK). Rabbit

108

monoclonal anti-Na+/K+-ATPase α1 antibody was from Cell Signaling Technology

109

(Boston, MA, USA). HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody

110

was from TDY Biotechnology (Beijing, China).

111

Quinine, aristolochic acid, D-(-)-salicin and platycodin D (purity ≥ 97%) were from

112

Nature Standard Technology (Shanghai, China). Platycodin L (purity ≥ 98%) were

113

from Quality Phytochemicals (New Jersey, USA). Denatonium benzoate, probenecid,

114

acid red 1, 3-mercaptopropionic acid (3-MPA), 1-ethyl-3-(3-dimethylaminopropyl)

115

carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (all purity ≥

116

98%) were from Sigma-Aldrich (St. Louis, MO, USA). Fluo-4 AM dye was from

117

Molecular Probes (Grand Island, NY, USA). Matrigel was from Becton Dickinson

118

(New York, NY, USA). Methanol, acetonitrile, formic acid (UPLC grade) and

119

dimethylsulfoxide (DMSO, HPLC grade) were from Honeywell (Morris, NJ, USA).

120

Deionized water was produced by the Millipore-Q water purification system

121

(Millipore, Bedford, MA, USA).

122

P. grandiflorum were collected from Anhui, China in May, 2016 (Batch No.

123

16052904) and identified by Prof. Zhenfang Bai (School of Chinese Materia Medica,

124

Beijing University of Chinese Medicine, Beijing, China). Voucher specimens were

125

preserved at the authors’ laboratory.

126

Establishment of Pharmacophore Model of TAS2R14 Agonists for Virtual 6


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Because of the unavailability of 3D crystal structure of TAS2R14, the

127

Screening

128

pharmacophore models of the TAS2R14 agonists were firstly established and

129

ligand-based virtual screening was adopted to predict the potential TAS2R14 agonists

130

in P. grandiflorum, which could reduce the blindness of the following experimental

131

procedure. Twelve known TAS2R14 agonists were used as a training set (Table S1) to

132

establish pharmacophore models22, taking into account the structural diversity and

133

activity of these compounds. The key steps for pharmacophore establishment were

134

briefly described as follows23. The 3D pharmacophore hypotheses were constructed

135

by HipHop (Common Feature Pharmacophore Generation) in Discovery Studio v4.0

136

(Accelrys, San Diego, CA, USA). Conformations of ligand were generated within the

137

relative energy threshold of 20 kcal/mol by BEST (Best Quality Conformer

138

Generation) at the maximum size of 255 conformations. On the basis of initial

139

analysis, hydrogen bond acceptors (HBA), hydrogen bond donors (HBD),

140

hydrophobic portions (HY) and aromatic rings (AR), which well-matched all of the

141

training set ligands have been selected. The interaction between ligand and receptor

142

could be characterized by these pharmacophore features. The maximum number of

143

pharmacophore models produced was set as 10. A decoy set, consisting of 37

144

experimentally known TAS2R14 agonists22 (Table S2) and 191 non-active compounds

145

(Table S3), was applied to validate the established pharmacophore models. An

146

empirical parameter CAI (comprehensive appraisal index) were used to quantify the

147

capabilities of pharmacophore models23. The one with the highest CAI was picked as

148

query to screen the self-built 3D chemical database including 121 reported 7



149

compounds derived from P. grandiflorum in Discovery Studio. The minimum

150

interference distance was set as 1 Å, and search algorithm was set as best. The other

151

parameters were set as default. Finally, the fit values were calculated to denote the

152

matching degree between the conformers of each compound and pharmacophore

153

model, namely a higher fit value indicated a better match.

154

Construction of Recombinational TAS2R14-HEK293 Cells and Extraction of To prepare the bioactive

155

Membrane Protein with Over-expressed TAS2R14

156

TAS2R14, a recombinational TAS2R14 (Gα16gust44)-HEK293 cell line was

157

constructed, in which TAS2R14 was over-expressed. The coexistence of Gα16 protein

158

and gustducin of 44 amino acids was necessary for testing TAS2R14-agonistic

159

activity in intracellular calcium mobilization assay24. A full-length human cDNA of

160

TAS2R14 was firstly cloned and then co-transfected with the gene of Gα16-gustducin

161

into HEK293 cells24. Then the recombinational TAS2R14-HEK293 cells were

162

incubated in 5% CO2 at 37 °C, and the medium was DMEM containing 10% FBS, 100

163

U/mL penicillin and 100 µg/mL streptomycin, 100 µg/mL hygromycin B and 200

164

µg/mL G418. The last two kinds of antibiotic were used to inhibit the growth of

165

non-transfected cells and consequently screen TAS2R14-HEK293 cells. Due to the

166

existence fluorescent tag within the constructed TAS2R14 expression vector, red

167

fluorescence from stably growing TAS2R14-HEK293 cells should be easily observed

168

under a fluorescence microscope (Figure S1). For comparison, primordial HEK293

169

cells were also cultured, but in DMEM just added 10% FBS, 100 U/mL penicillin and

170

100 µg/mL streptomycin. After culturing and harvesting sufficient cells, membrane 8


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171

fractions were extracted using Mem-PER™ plus membrane protein extraction kit

172

according to the suggested protocol.

173

Western Blotting for Identification and Quantification of TAS2R14

The

174

concentration of extracted membrane protein was determined using the BCA kit. Then

175

Western

176

TAS2R14-HEK293 cells and comparing its content in membrane protein extracted

177

from TAS2R14-HEK293 cells and HEK293 cells. Na+/K+-ATPase α1 was used as the

178

internal inference. The membrane fractions were firstly separated by SDS-PAGE and

179

transferred to nitrocellulose membranes. After blocking with 3% BSA-TBST for 30

180

min, the membranes were incubated overnight in primary antibodies at 4 °C. The

181

primary antibodies were rabbit polyclonal anti-TAS2R14 antibody and rabbit

182

monoclonal anti-Na+/K+-ATPase α1 antibody (both 1:5000 diluted with 3%

183

BSA-TBST). After incubation, the membranes were exposed to HRP-conjugated goat

184

anti-rabbit IgG (H+L) secondary antibody (1:2000 diluted with 5% nonfat milk-TBST)

185

for 40 min. Finally, the membranes were processed using the ChemistarTM High-sig

186

ECL Western blotting substrate (Tanon, Shanghai, China) and analyzed on a GeneTex

187

image analyzer (Fujifilm, Tokyo, Japan).

188

blotting

was

adopted

to

identify

Fabrication of AlGaAs/InGaAs HEMT Sensor

TAS2R14

in

recombinant

The AlGaAs/InGaAs HEMT were

189

grown by molecular beam epitaxy (MBE) on GaAs substrate25. From bottom to top, it

190

consisted of an undoped GaAs buffer layer (500 nm), an In0.3Ga0.7As channel layer

191

(15 nm), an Al0.3Ga0.7As spacer layer (4 nm), a Si δ-doping layer, an Al0.3Ga0.7As

192

barrier layer (25 nm) and a GaAs cap layers (30 nm). Figure 1 shows the 9



193

cross-sectional schematic drawing of AlGaAs/InGaAs HEMT device. The detailed

194

fabrication process of HEMT was described as the reference26.

195

Immobilization of TAS2R14 on AlGaAs/InGaAs HEMT Sensor and XPS

196

Characterization Before the bio-functionalization process, a quartz glass tube (10 ×

197

4 mm) was stuck on the surface of chip with “502” glue carefully, which was used as

198

a solution reservoir. The chip consisted of four groups of HEMT with 4 × 2 Au-coated

199

gates just surrounded in the tube (Figure 1). 10 mM 3-MPA aqueous solution was then

200

added into the reservoir at room temperature for 24 h to form the self-assembled

201

monolayer (SAM). After Au-S bonding, the sensor was washed with water to remove

202

3-MPA physically absorbed. Subsequently, a carboxyl activating aqueous solution

203

containing equivoluminal 20 mM EDC and 50 mM NHS was dropped into the

204

reservoir, reacted at room temperature for 15 min and rinsed with 100 mM PBS. The

205

activation reaction produced stable amine adducts27. Next, the extracted membrane

206

protein containing over-expressed TAS2R14 was dissolved in 100 mM PBS (200

207

µg/mL) and introduced into the reservoir and incubated at 4 °C for 2 h followed by

208

washing.

209

XPS were carried out to characterize the immobilization of protein on Au-deposited

210

GaAs substrates. XPS spectra were acquired by a K-Alpha XPS system (Thermo

211

Fisher Scientific, USA) equipped with a monochromatic Al Kα source and a

212

hemispherical electron energy analyzer. Low-resolution full scans (1 eV/step) and

213

high-resolution fine scans (0.05 eV/step) of C1s, N1s and S2p were obtained,

214

respectively. The software Avantage v5.965 (Thermo Fisher Scientific, USA) were 10


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employed to process XPS data. Verification of Sensitivity and Response Range of TAS2R14-functionalized HEMT To determine the sensitivity and response range of TAS2R14-functionalized

217

Sensor

218

HEMT sensor, the tests were performed using quinine solution of 11 concentration

219

gradients ranging from 0.1 pM to 1 mM as samples, and the HEMT sensor modified

220

using membrane protein without over-expressed TAS2R14 was used as negative

221

control. Quinine was dissolved with 5% DMSO aqueous solution, which could fully

222

dissolve the drug and minimize the damage to the activity of protein. The solvent was

223

also used as the blank control throughout the experiment. Three minutes after adding

224

drug solution into the reservoir, the measurement process was carried out on the probe

225

platform. The drain-source current (IDS) versus voltage (VDS) of the biosensor was

226

acquired at room temperature by a CHI-660E Electrochemical Workstation (Huake

227

Putian Technology, Beijing, China). According to previous experience, the biosensor

228

would be broken down when VDS exceeded 2 V, so the IDS were measured in the VDS

229

scope of 0 to 2 V. Each test was repeated three times at each concentration. The IDS at

230

VDS equal to 1 V was selected to calculate the realtive IDS and plot the

231

response-concertration corelation.

232

Verification of Specificity of TAS2R14-functionalized HEMT Sensor Quine and

233

aristolochic acid were two known TAS2R14 agonists22, while D-(-)-salicin and

234

denatonium

235

TAS2R14-functionalized HEMT sensor was exposed to solutions of these four bitter

236

substances at 1 nM individually to further verify its specificity, and the HEMT sensor

benzoate

were

non-agonists8.

In

11


this

investigation,

the


237

modified using membrane protein without over-expressed TAS2R14 was used as

238

negative control. All the drugs for test were also dissolved with 5% DMSO aqueous

239

solution. After 3 min reaction, IDS versus VDS were recorded in the scope of 0 to 2 V at

240

room temperature, and every test was repeated three times. The IDS at VDS equal to 1 V

241

was selected to calculate the relative IDS.

242

Screening of Potential TAS2R14 Agonists from P. grandiflorum Using Five hundred milligrams air-dried and

243

TAS2R14-functionalized HEMT Sensor

244

ground roots of P. grandiflorum were extracted by ultrasonic-assisted method with 25

245

mL methanol for 30 min. The extracts was filtered and dried in vacuum, then

246

redissolved in 25 mL 5% DMSO aqueous solution and filtered through a 0.22 µm

247

membrane as the sample.

248

Sixty microliters blank solvent and P. grandiflorum extracts sample were injected

249

to the reservoir of TAS2R14-functionalized HEMT sensor, respectively, and incubated

250

at room temperature for 3 min, followed by scanning the IDS-VDS curve. After

251

confirming the effective response derived from the bound ligands, the biosensor was

252

washed with 60 µL non-specific eluent agent (100 mM PBS of pH7.4) three times to

253

remove any unbound components, and then treated with 60 µL specific eluent agent (1

254

µM quinine) for 3 min to release the captured potential TAS2R14 agonists. The

255

released components were collected and then applied to UPLC-QTOF MS analysis

256

for peak identification.

257

UPLC-QTOF MS Analysis and Identification for Potential TAS2R14 Agonists The

258

components eluted from the biosensor was analyzed on Agilent 1260 Infinity UPLC 12


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259

coupled with Agilent 6540 Exact Mass QTOF MS system (Agilent, Santa Clara, CA,

260

USA). The column used for separation was an Agilent Zorbax RRHD Ecipse Plus C18

261

column (100 × 3.0 mm, 1.8 µm; Agilent, USA), which performed at 40 °C. The

262

mobile phase consisted of solvent A (0.5% formic acid in water, v/v) and solvent B

263

(acetonitrile) using a gradient elution program as follows: 30–50% B at 0–5 min. The

264

flow rate was kept at 0.4 mL/min. The sample volume injected was set at 2 µL. Mass

265

analysis was carried out in negative ESI mode, and the parameters of ion source were

266

set as follows: drying gas temperature, 200 °C; drying gas flow rate, 11 L/min;

267

nebulizer pressure, 35 psig; sheath gas flow rate, 9 L/min; sheath gas temperature,

268

225 °C; capillary voltage, 3 500 V; nozzle voltage, 1,000 V; fragmentor voltage, 380

269

V and Oct 1 RF Vpp, 750 V. The acquired mass range was set at m/z 100 – 1,500 Da.

270

All other MS parameters were left as default settings. The software Agilent

271

MassHunter Qualitative Analysis B07.00 (Agilent, USA) were applied for MS data

272

processing. The components identification was conducted through the process of

273

generating molecular formulas on the basis of m/z values of quasi-molecular ions,

274

searching reported compounds in the genus of Platycodon and confirming targeted

275

structures. The threshold of mass error was fixed at 3 ppm.

276

Evalution for Screened TAS2R14 Agonists by Intracellular Calcium Mobilization

277

Assay The intracellular calcium mobilization assay was employed to evaluate the

278

TAS2R14-agonistic activity of those potential agonists. However, due to the

279

unavailability of standard substances of the other four compounds, only platycodin D

280

and platycodin L were applied in this assay. The two different drugs were dissolved in 13



281

DMSO at 100 mM. And 8 µM aristolochic acid and 0.25% DMSO were used as the

282

positive and negative control, respectively. The final concentration of DMSO in each

283

well did not exceed 0.25% for all the tested drugs. The TAS2R14-HEK293 cells were

284

seeded into 96-well plates coated with Matrigel at a density of 3.0 × 104/well and

285

cultured in 5% CO2 at 37 °C overnight. Before assay, the medium in each well was

286

substituted by 100 µL of loading buffer containing 4 µM Ca2+-sensitive Fluo-4 AM

287

dye, 2.5 mM probenecid and 2 mM acid red 1 in HBSS. The plate was then incubated

288

in dark for 30 min before detecting the calcium signal. For studying the antagonism,

289

80 µL loading buffer was complemented to each well, together with 20 µL HBSS

290

containing tested drugs with suitable concentration 10 min before calcium-flux

291

detection. Cells were transferred to a FlexStation Ⅲ (Molecular Devices, San Jose,

292

CA, USA) for fluorescence scanning. Basal fluorescence was scanned for 16 s prior to

293

the agonist was applied. Then the integrated fluidics system of FlexStation Ⅲ

294

transferred 25 µL drug solution from the drug plate to assay plate, which contained

295

100 µL of loading buffer. The relative fluorescence units (RFU) were recorded at

296

37 °C at an excitation wavelength of 485 nm and emission wavelength of 525 nm,

297

every 2 s for 100 s. At last, the EC50 value was calculated by RFU. Each data

298

represents the mean ± standard deviation in six replicates.

299

A specificity assay was performed to eliminate the false positive results in activity

300

evaluation. If a drug exhibited equivalent ability to induce intracellular calcium-influx

301

in both HEK293 cells and TAS2R14-HEK293 cells, it would be thought as the false

302

positive. Herein, HEK293 cells were treated with platycodin D and platycodin L of 14


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303

various concentrations to observe whether they could induce calcium-influx. The

304

positive and negative controls were same as to the above assay.

305

Statistical Tests

All data acquired in performance characterization of biosensor

306

and intracellular calcium mobilization assay were processed using one-way analysis

307

of variance (ANOVA) followed by Student’s t-test to find the differences between

308

group means in GraphPad Prism v7.0 (GraphPad Software, La Jolla, CA, USA); p

Discovery of TAS2R14 Agonists from Platycodon grandiflorum Using

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