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Discovery of 4-arylthiophene-3-carboxylic acid as inhibitor of ANO1 and its effect as analgesic agent

Anoctamin 1 (ANO1) is a kind of calcium-activated chloride channel involved in nerve depolarization. ANO1 inhibitors display significant analgesic activity by the local peripheral and intrathecal administration. In this study, several thiophenecarboxylic acid and benzoic acid derivatives were identified as novel ANO1 inhibitors through the shape-based virtual screening, among which the 4-arylthiophene-3-carboxylic acid analogues with the best ANO1 inhibitory activity were designed, synthesized and compound 42 (IC50 = 0.79 μmol/L) was finally obtained. Compound 42 selectively inhibited ANO1 without affecting ANO2 and intracellular Ca2+ concentration. Subsequently, the analgesic effect was investigated by intragastric administration in pain models. Compound 42 significantly attenuated allodynia which was induced by formalin and chronic constriction injury. Through homology modeling and molecular dynamics, the binding site was predicted to be located near the calcium-binding region between α6 and α8. Our study validates ANO1 inhibitors having a significant analgesic effect by intragastric administration and also provides selective molecular tools for ANO1-related research.


Introduction
Anoctamin 1 (ANO1, TMEM16A) is a kind of the calciumactivated chloride channel (CaCC) on cytomembrane, controlled by intracellular Ca 2þ concentration and membrane potential 1e8 . ANO1 is mainly expressed in airway 9,10 , peripheral nervous system 11 , gastrointestinal smooth muscle 12 , and some tumors 13e15 , which mediates and controls anion permeation to fulfill airway and exocrine gland secretion, neuronal signal transduction, and rhythmic movements of the gastrointestinal system.
In the peripheral nervous system, ANO1 is distributed in nociceptors, dorsal root ganglia (DRG), and spinal nerves, involved in nerve depolarization 11,16,17 . ANO1 is capable of augmenting the excitability of DRG neurons under inflammatory or neuropathic conditions and thereby aggravates inflammation-or tissue injuryinduced pathological pain 18,19 . Knockout or inhibition of ANO1 causes significant analgesic effects. Intrathecal injection of the ANO1 inhibitor reverts the spinal nerve ligation-induced mechanical allodynia in a dose-dependent manner 16 . The non-selective and selective CaCC blockers reduce formalin-induced flinching behavior mainly during phase 2 of the formalin test 20 .
ANO1 is considered as associated with inflammation-related pain caused by the changes in temperature, ion concentration in inflamed tissues, and inflammatory factors 21 . The elevated temperature of inflamed tissue activates temperature-sensitive calcium channels (such as transient receptor potential channel V1, TRPV1), which activates ANO1 not only by direct interaction 21,22 but also indirectly by increasing intracellular calcium ion concentrations 23 . Interleukin-4, an inflammatory factor in inflamed tissues or injured nerves, can promote the overexpression of ANO1, which may be related to allodynia 5,16 .
In this study, to explore new ANO1 inhibitors and potential analgesic agents, we firstly performed a scaffold virtual screening starting from the 2-arylamidothiophene skeleton shared in CaCC inh -A01 (1) and AACT (4), and a series of thiophenic acid and benzoic acid derivatives showed ANO1 inhibition. Subsequently, the 4-arylthiophene-3-acid selected from the screening results was used as the starting hit for optimization. The inhibitory activities were evaluated by whole-cell patch clamp on ANO1-overexpressing Fischer rat thyroid (FRT) cells. The activity preferred compound was used for analgesic evaluation in various animal pain models. The finding of this study is helpful for the understanding of the structureeactivity relationships (SARs) of ANO1 inhibitors and provides a selective inhibitor for probing the biological function of the ANO1 channel.

Shape and electrostatic similarity-based virtual screening
The shape and electrostatic similarity-based virtual screening query were generated from the optimized conformation of CaCC inh -A01 in OPENEYE software (Fig. 2). 300,000 compounds from the SPECS chemical library and Chinese National Compound Library of Peking University (PKU_CNCL) were screened followed by artificial selection of the molecules with Shape Tanimoto and EON index greater than 0.6. Seventy-six compounds, including thiophenic acid derivatives (Table 1, compounds 7e32), benzoic acid derivatives (Supporting Information Fig. S1, compounds S1eS13), and 37 other scaffold compounds (Fig. S1, compounds S14eS50). Their ANO1 inhibitory effects were tested using whole cell patch clamp at 100 mmol/L in ANO1-overexpression FRT cell line.
Of these 76 structurally diversified compounds, we focused on three thiophene containing scaffolds (scaffolds 1e3) which CaCC inh -A01 and AACT share (compounds 7e32, Table 1), while the ANO1 inhibitory activity of other compounds was shown in Supporting Information Table S1 due to they showed relatively low ANO1 inhibitory activity in whole-cell patch clamp.
The scaffold 1 varies in the ring size of tetrahydrobenzo[b] thiophene in CaCC inh -A01 (1). Active compounds appeared to have t-butyl substitution as R 2 (1 and 7). Compounds with 2-Fphenyl (10), 4-I-phenyl (12), and 1-naphthalene (14) as R 1 were more active than others. Extending the amide linker by the double Figure 1 Chemical structure of reported ANO1 inhibitors. bond (15) or methylene (16) is detrimental to the ANO1 inhibitory activity. Expanding the thiophene-fused aliphatic ring contributes to the enhancement of ANO1 inhibitory activity, for example, compound 17 (n Z 3) is better than compound 9 (n Z 2).
In scaffold 2, the moiety of tetrahydrobenzothiophene is replaced by thiophene in which positions 4 0 and 5 0 are substituted by alkyl or aromatic groups. Compounds 22 and 23 display comparative ANO1 inhibitory activity with CaCC inh -A01 (1), indicating the feasibility of breaking tetrahydrobenzo-ring. Alkyl substitution at R 2 (22 and 23) is more favorable than nonsubstitution (21). Among the substituents of the R 1 , 4methylphenyl (22) is superior to 4-tert-butylphenyl (24) and 3bromophenyl (25). Extending the amide linker by the double bond is unfavorable to the ANO1 inhibitory activity.
Based on the preliminary SAR analysis, compound 28 with 4arylthiophene-3-carboxylic acid backbone was selected as a hit for further optimization.

Synthesis and optimization of 4-arylthiophene-3-carboxylic acid derivatives
Based on compound 28, further optimization strategies involved three steps: firstly, 4-methoxyphenyl at thiophene 4 0 position was replaced by various substituted phenyl and naphthyl groups. Then, the substructure on the carbonyl side of the amide was replaced by the preferred groups from the above virtual screening process. Finally, combined the substructures of tetrahydrobenzothiophene in CaCC inh -A01 and the aryl substituted thiophene at 4 0 position in compound 28 to expand the volume of thiophene containing scaffold. ANO1 inhibitory rate was conducted by yellow fluorescence protein (YFP) fluorescence quenching assay 24 in ANO1overexpressing FRT cell line and was calculate by Eq. (1): where K cont means the slope of negative control, K comp means the slope of the test compound. The ANO1 inhibitory IC 50 was determined based on the inhibitory rate in 7 compound concentrations. Compound 42 and its derivatives were synthesized by three steps (Schemes 1 and 2). Firstly, ketones reacted with cyanoacetate and sulfur to form 2-aminothiophene-3-carboxylate acid ethyl ester intermediates by the Gewald reaction 33 . Then these intermediates are coupled with acyl chloride to form the amides derivatives. Finally, the hydrolysis reactions were conducted to form final products.
Compounds 47e53 combined the substructures of tetrahydrobenzothiophene in CaCC inh -A01 and the aryl substituted thiophene at 4 0 position in scaffold 3 (Table 3). When the R 1 was H (47), the inhibitory activity against ANO1 was higher than that of the methoxy group (48). Methyl substitution at R 2 and R 3 positions (42) favored ANO1 inhibitory activity. 1-Naphthyl (51) or 2naphthyl (50) at R 4 position was beneficial to ANO1 inhibition. Compound 53 was more active than compound 47, showed that the thiophene-fused a larger cycloalkane had a higher ANO1 inhibitory activity. It indicated that the flexibility between thiophene and benzene rings might favor activity. However, compounds 47e53 showed poor solubility in synthesis (only completely soluble in DMSO, slightly soluble in methanol, insoluble in water and DCM), which would cause a lot of trouble for further study, so we terminated the optimization of these scaffolds.
Based on the SAR studies of thiophene-containing ANO1 inhibitors (both in virtual screening and structure optimization), compound 42 (IC 50 Z 0.79 mmol/L) was finally selected for further pharmacokinetic and pharmacodynamics study.

Electrophysiology and Ca 2þ fluorescence investigations of compound 42
To verify the inhibitory activity of compound 42 on ANO1, we performed whole-cell patch clamp in ANO1-overexpressing FRT cells as previously described 10 . In this experiment, the ANO1 channel was activated by 1 mmol/L intracellular Ca 2þ followed by changing the membrane potential þ100 to À100 mV and comparing the evoked CaCC current before and after treatment. As shown in Fig. 3A, 30 mmol/L compound 42 significantly suppressed the CaCC current and was more potent than Ani9. ANO1 is activated by intracellular Ca 2þ , and if the compound inhibits the Ca 2þ transmembrane, it will aggravate its ANO1 inhibitory effect. Calcium fluorescent dye Cal520™ was used to evaluate the influence of compound 42 on intracellular Ca 2þ concentration. Amlodipine was selected as a positive control. After 2 h pretreatment with Cal520™, compound 42 and ATP were added in turn to evoke the Ca 2þ influx across cytomembrane, which showed the maximum relative fluorescence units (RFU) of the control group increased to 2.3 folds of the initial value and the RFU of amlodipine remained unchanged ( Fig. 3B and C). Compound 42 almost did not affect Ca 2þ influx (1.9 folds than initial value), and CaCC inh -A01 slightly suppressed Ca 2þ influx (1.5 folds than initial value), whereas Ani9 hinders Ca 2þ transmembrane.

ANO1/ANO2 channels selectivity of compound 42
ANO2 coexists with BK (large conductance calcium-activated potassium) and SK (small conductance calcium-activated potassium) channels in inferior olivary neurons that participate in the   3 13.0 a ANO1 inhibition rate of compounds at 100 mmol/L was determined using whole-cell patch clamp on ANO1-overexpressing FRT cells at þ100 mV voltage. Data are represented by mean AE SEM, n Z 5.
control of motor learning and timing 34 . ANO1-targeting analgesics lacking ANO1/ANO2 selectivity may cause potential central nervous system adverse effects. Thus, the ANO1/ANO2 selectivity of compound 42 was evaluated. ANO2 gene was transiently transfected into HEK293 cell, and then ANO2-mediated CaCC current was recorded by whole-cell patch clamp. The detailed method is the same as the reference 35 . The ANO1 and ANO2 paninhibitor CaCC inh -A01 was used as a negative control, and ANO1 selective inhibitor Ani9 was applied to be a positive control. The results showed that compound 42 at 30 mmol/L had little effect on    ANO2 induced CaCC current. The ANO1/ANO2 selectivity of compound 42 is slightly weaker than that of Ani9 but much higher than that of CaCC inh -A01 (Fig. 4).

Stability and MDCK permeability of compound 42
We wanted to test the in vivo analgesic activity of ANO1 inhibitors by intragastric gavage (i.g.) administration rather than local peripheral and intrathecal administration as previously reported 16,19,20 , so compound 42 was evaluated for acid-base stability and intestinal absorbability to ensure it could be absorbed by animals.

Stability of compound 42 under different pH condition
The acid-base stability of compounds was tested under different pH buffer incubation by HPLC, which showed compound 42 remained stable in both acidic and alkaline environments (Table 4).   (Fig. 5). All of the testing ANO1 inhibitors displayed proliferation inhibitory activity at 10 mmol/L or higher. Compound 42 and T16A inh -A01 had a weak effect on proliferation under 1 and 3 mmol/L, while CaCC inh -A01 inhibited cell proliferation at all concentrations tested.

Acute toxicity of compound 42
The acute toxicity was carried out in 6e8 weeks aged C57BL/6 male mice which were treated with 1000 mg/kg compound 42 by i.g. administration. All mice survived and no intense acute toxic response (such as coma, tachypnea, and moribund status) was observed in 72 h. The acute toxicity test showed that compound 42 is safe at the dose of 1000 mg/kg or lower by i.g. administration.

In vivo analgesic effects of compound 42
The preliminary in vivo analgesic effects of compound 42 were investigated in the formalin test (Fig. 6A), chronic constriction injury model (Fig. 6B), hot plate test (Supporting Information Fig. S3) and writhing test (Supporting Information Fig. S4).
In the formalin test, phase 1 of the nociceptive response began immediately after formalin administration and then declined gradually in approximately 10 min. Phase 2 began about 15 min after formalin administration and lasted about 1 h. Pregabalin and flupirtine were selected as positive compounds. The results showed that these compounds had no significant effect on the number of foot lifts compared with the vehicle group (Fig. 6A). While, it was very encouraging that 80 mg/kg compound 42 (70 foot lifts) showed a significant analgesic effect in phase 2 compared with vehicle (121 foot lifts), which was almost as effective as flupirtine at 200 mg/kg (74 foot lifts) and pregabalin at 40 mg/kg (44 foot lifts). CaCC inh -A01 at 80 mg/kg (84 foot lifts) had no significant analgesic effect. Compound 42, as an ANO1 inhibitor, has a significant analgesic effect on phase 2 but not phase 1 in formalin test, which is consistent with the previous reports 20 .
The chronic constriction injury (CCI) of the sciatic nerve in the rat was used to evaluate the analgesic effects of compounds on the F prediction, oral bioavailability prediction; data are represented by mean AE SD, n Z 3.

Figure 5
Proliferation of CHO cells incubated with ANO1 inhibitors. Bar graph represents cytotoxicity of ANO1 inhibitors on CHO cell proliferation; n Z 3; data are represented by mean AE SEM; NS, no significant, *P < 0.05, **P < 0.01, ***P < 0.001 compared to the 0.1% (v/v) DMSO in each group (control), t-test. Figure 6 The results of in vivo analgesic test. (A) Formalin test on rat; n Z 12; the control group was injected with 50 mL saline instead of 5% (v/v) formalin. (B) Chronic constriction injury model (CCI) on rat; n Z 8. Basic represents basic pain threshold before sciatic nerve ligation operation; 0 h, before corresponding compounds treated; 1 h, 1 h after the corresponding compound treated. PTI, the pain threshold rate change (%) of 1 h compared to 0 h. mpk represents mg/kg. All data in (A) and (B) are represented by the mean AE SD. Statistical significance was determined by ANOVA, *P < 0.05 and **P < 0.01 compared to the vehicle. nerve injure-derived hyperalgesia. As shown in Fig. 6B, the threshold of paw withdraw measured by Electronic Von Frey before (basic) and after (0 h) CCI model established, and 1 h after compounds administration the threshold of paw withdraw was measured again (1 h). Pregabalin (40 mg/kg) and flupirtine (100 mg/kg) could significantly increase the threshold of paw withdraw with the pain threshold rate change (PTI, %) of 60.4 and 36.2 respectively. Treatment with compound 42 at 40 mg/kg also resulted in a significant analgesic effect (PTI of 45.2), which was more potent than 100 mg/kg flupirtine.
Based on the analgesic effects of ANO1 inhibitor compound 42 in different kinds of pain models, by i.g. administration compound 42 displayed more strength analgesic effects on chronic pain, including formalin-induced allodynia and sciatic nerve ligation-induced mechanical allodynia, especially on inflammation-related chronic pain (phase 2 in formalin test). For acute pain, however, no significant analgesic effect was observed with ANO1 inhibitors treatment in the hot plate test and the writhing test (Supporting Information Figs. S3 and S4).

Binding pose prediction of compound 42
To get insight into the binding behavior of ANO1 and its inhibitor, a lot of computer-aided drug design work has been conducted. Firstly, the human ANO1 (hANO1) homology modeling of dimer structure (Fig. 7A, Supporting Information) was constructed based on mANO1 cryo-electron microscopy structure 36e40 using Schrödinger Prime software (sequence alignment results are shown in Supporting Information Fig. S5). Then Schrödinger SiteMap software was used to predict binding sites, and three ligand binding sites were provided (Fig. 7B). Site 1 is located outside the membrane surrounded by the terminals of a5, a6, and a9. Site 2 is adjacent to the Ca 2þ binding region and consists of a6, a7, and a8. Site 3 was near the exit of the pore inside the membrane.
Many hydrophilic arginine and glutamine residues enriched in site 3, lead to most of the hydrophobic ANO1 inhibitors were incapable to bind this area, so site 3 was ruled out first. Then reported ANO1 inhibitors (Fig. 1) and compound 42 were docked into site 1 and site 2 to evaluate whether the docking conformation conformed to the corresponding SAR. In site 1, ANO1 inhibitors docking conformation were randomly distributed in the binding pocket around K865, which indicated site 1 volume is too large for these inhibitors to form a stable docking conformation (Fig. 7C). In site 2, all inhibitors showed a very consistent binding conformation (Fig. 7D): the aromatic ring on the carbonyl side of the amide bond was embedded in the hydrophobic loop between a7 and a8, the carboxylic acid kept towards K767. As previously reported 39,40 , a6 and the calcium-binding region produced large conformational changes during Ca 2þ induced ANO1 activation, thereby compounds binding to this region might be effective to inhibit ANO1. Thereby, site 2 is more likely to be the inhibitors binding site. Flexible molecular docking was used to dock the CaCC inh -A01, AACT, and compound 42 into the site 2, and 30 ns molecular dynamic simulation was conducted based on docking conformation for more accurate protein-ligand interaction pattern (Fig. 8).
The carboxyl group of compound 42 and CaCC inh -A01 formed an electrostatic interaction with K767. Amide in all three compounds formed hydrogen bonds to A723 and G724 main chain. Naphthalene of compound 42 is embedded into the hydrophobic loop between a7 and a8 to form the cation-p interaction with K767. The 4-chlorophenyl of compound 42 formed the cation-p interaction with K614.
To verify the binding site, we mutated the hANO1 residues and constructed a transient ANO1 high expression cell line on HEK293 cells. The differences of inhibition rate of 30 mmol/L CaCC inh -A01 against wild type (69.4 AE 9.8%, average AE SD, n Z 3) and mutant types were tested in whole-cell patch clamp at þ100 mV potential. R321A (in site 3) and K614A (in site 2) mutations did not affect ANO1 inhibitory activity of CaCC inh -A01, and K671A (near the Ca 2þ binding pocket) reduced ANO1 inhibition rate of CaCC inh -A01 (38.0 AE 13.6%, average AE SD, n Z 3, P < 0.05, t-test). Whereas the mutation of K687A, K767A, and F768A in site 2 changed the electrophysiological features of CaCC current, led to unable to test ANO1 inhibition. Therefore, the uncertainty still exists to reveal the binding site without other experimental evidence such as structural biology.

Conclusions
Herein, we found a series of thiophenic acid and benzoic acid derivative ANO1 inhibitors by virtual screening. Among which, 4arylthiophene-3-carboxylic acid scaffold was selected as a hit for further structural optimization, and compound 42 (IC 50 Z 790 nmol/L) was obtained. Activity verification showed that compound 42 could strongly inhibit the CaCC current in whole-cell patch clamp, and did not affect ANO2-induced CaCC current and weakly hinder Ca 2þ transmembrane. Furthermore, the preliminary analgesic activity of compound 42 was evaluated in a variety of pain models by i.g. administration. The results showed that compound 42 had significant analgesic activity on inflammation-related pain. Finally, the binding site of compound 42 to ANO1 was predicted by homology modeling, docking, and molecular dynamics, which suggested that the binding site was close to the Ca 2þ binding region. Our study is the first to show that an oral ANO1 inhibitor has a significant analgesic effect and also provides selective molecular tools for ANO1-related research.

Shape and electronic distribution-based virtual screening
Shape and electronic distribution-based virtual screening were based on CaCC inh -A01 optimized conformation using ROCs and EON in OPENEYE software. In ROCs, the parameters were set as: "Tanimoto cutoff 0.6, rank by Shape Tanimoto" and in EON, the parameters were set as default.

Chemistry
The screened compounds were purchased from the SPECS database and PKU-CNCL. The purities of all purchased compounds were more than 90%. For the chemical synthesis, reagents and solvents were obtained from J&K Chemical (Beijing, China) without further purification. T16A inh -A01 was purchased from TOCRIS (USA). 1 H-NMR and 13 C-NMR spectra were recorded on Bruker (400 MHz) instruments, using DMSO-d 6 or CDCl 3 as solvents. High-resolution mass spectra (HRMS) were recorded on the Bruker Apex IV FTMS mass spectrometer using ESI (electrospray ionization). The microwave-assisted reaction was performed on the CEM Discover SP microwave reactor. The purity of the final compound was determined by NMR and HPLC to be >95% (UV detection (l Z 254 nm), compound structures in smiles format and more detail can be found in Supporting Information).

Synthesis of compounds 33-46
Taking compound 42 as an example: 4-chloroacetophenone 18.55 g (120 mmol), ethyl cyanoacetate 16.29 g (144 mmol), acetic acid 7.12 g (120 mmol), morpholine 23.00 g (264 mmol), and ethanol 500 mL were added to a 1 L round bottom flask. Stirred at 68 C for 3 h under an argon atmosphere, followed by the addition of 4.91 g (144 mmol) sulfur and then stirred for 48 h. The solution was diluted with DCM and washed with brine four times. The organic phase was separated and evaporated 10 mL EA was added to re-dissolve the residue. Acidic EA was added, the precipitates were collected, washed, and then was added to 200 mL 50% ammonia and stirred for 30 min. Followed by extraction with DCM four times, the combined organic phase dried with anhydrous Na 2 SO 4 . After evaporation, 2aminothiophene-3-carboxylate acid ethyl ester was obtained (yield 21%). 0.195 g (0.73 mmol) 2-aminothiophene-3carboxylate acid ethyl ester was dissolved in 10 mL DCM, and 0.125 g (0.66 mmol) 1-naphthyl chloride in 10 mL DCM was added at 0 C. The solution was stirred at room temperature for 8 h, followed by evaporation and recrystallization using DCM/ petroleum ether. White powder intermediate 0.227 g (0.54 mmol) was obtained (yield 75%). 0.143 g (0.34 mmol) white powder intermediate and NaOH 0.068 g (1.7 mmol) were dissolved in THF/methanol/water solution and stirred at room temperature for 8 h, acetic acid was used to adjust pH to neutral and water was added to precipitate. 0.094 g (0.23 mmol) 42 was obtained at a yield of 69%.

Synthesis of compounds 47-53
Taking compound 48 as an example. Indanone 1.409 g (8.0 mmol), ethyl cyanoacetate 1.352 g (9.6 mmol), acetic acid 0.480 g (9.6 mmol), ammonium acetate 0.736 g (9.6 mmol), and toluene 100 mL were added into a round bottom flask and refluxed for 8 h. After evaporation, morpholine 0.832 g (76.8 mmol), sulfur 0.384 g (9.6 mmol), and ethanol 100 mL were added and stirred for 48 h at 80 C under an argon atmosphere. The reaction solution was diluted with 150 mL DCM, washed with brine four times, and dried with anhydrous Na 2 SO 4 . After evaporation and column chromatography purification, brown solid immediate was obtained (yield, 35%). The following procedures were the same as compound 42. Finally, the brown powder compound 48 was obtained.

Electrophysiology
ANO2 currents were recorded from whole cell patches at room temperature from ANO2 transiently expressing HEK293 cell line. The protocol, pipet solution, and bath solution are the same as the reference 35 . HEK293 cells were depolarized from the holding potential of À40 mV to test potentials (þ100 to À80 mV) by þ20 mV increment for 500 ms and subsequently repolarized to À80 mV for 250 ms every 15 s.

YFP fluorescence quenching assay
The method of YFP fluorescence quenching assay is similar to the reference 10,41 . The ANO1-overexpressed FRT cell line was plated in 96-well plates and washed with PBS three times, and incubated with 60 mL test compound/PBS solution for 1 h (PBS with 0.1% (v/v) DMSO as a negative control). The fluorescence was measured in Microplate Reader (Flexstation 3, Molecular Devices) at a frequency of once per 1.7 s for 120 s (l ex /l em : 514 nm/ 535 nm). During the measurement, baseline fluorescence data F 0 was collected for 17 s before the addition of a PBS-iodide solution (PBS with 100 mmol/L NaI) containing 200 mmol/L ATP. FluorescenceÀtime curve was fitted by Eq. (2): The slope was calculated from 5 to 6 points where the fluorescence starts to decrease after adding the PBS-iodide solution. The inhibition rate was calculated by Eq. (1) which is described in Section 2.2. IC 50 was calculated from the inhibitione concentration curve fitted by the Hill equation. Data regulation and calculation were performed with Origin 8 software. Inhibition rateeconcentration curves of compounds are in Supporting Information Fig. S2.

Intracellular Ca 2þ fluorescence
HEK-293T cells were plated in 96-well plates and cultured for 24 h before changing the medium of each well into 100 mL DMEM with 10% (w/v) F127 and 10 mmol/L cal520™. After 2 h incubation, the cells were washed with PBS and incubated for 0.5 h with 100 mL compound/PBS solution at 30 mmol/L compound concentration. Cells were imaged before and 5 s after the addition of 200 mmol/L ATP containing PBS under Nikon eclipse Ti-S inverted fluorescence microscope with the FITC filter, and the data were analyzed with ImageJ software.

Stability under different pH conditions
The target compounds were diluted to 100 mmol/L with pH 1.0, 4.0, 6.8, 7.4, and 9.0 buffers, and the 0 h peak area of the target compounds were obtained by HPLCÀUV analysis. Then each pH compound solution was placed in 37 C water bath. The peak areas of the target compounds were obtained at 2 and 5 h. The change rate in the peak area was obtained by comparing the peak area of the target compound at different times with the peak area of 0 h. pH phosphate buffers were purchased from Shanghai Rongbai Biological Technology Ltd. (Shanghai, China).

MDCK cells permeability
A resistance-stable MDCK monolayer cell model was established in the transwell. A 100 mL compound solution was added to the upper chamber of the transwell, and 0.6 mL HBSS was added to the lower chamber, followed by incubation for 120 min at 37 C and 5% CO 2 . Then the lower chamber liquid was detected by LCÀMS (G6120B, Agilent Technologies, USA), and the compound concentration was calculated. The apparent permeability coefficient was calculated according to Eq. (3): DQ (mg) is the compound transport amount in Dt (s), A (cm 2 ) is the membrane area, and C 0 (mg/mL) is the initial drug concentration in the upper chamber of MDCK cells.

Cytotoxicity
The effect of ANO1 inhibitors on cell viability was determined by the SRB (sulforhodamine B) assay. Briefly, cells were cultured on sterile slides in 96-well plates overnight at 37 C with 5% CO 2 . Pregabalin, flupirtine, and morphine hydrochloride injection were obtained from Jiangsu Ehwa Pharmaceutical Group Co., Ltd. PEG400 was purchased from Weier Huagong (Nanjing, China). Administration volume was 1 mL/100 g mouse or rat weight. Most of the compounds in pharmacodynamics tests were conducted by i.g. administration, except for morphine by intraperitoneal (i.p.) administration. Formulation (take 80 mg/kg as example): 80 mg compound 42 sodium salt was dissolved in 2.0 mL DMSO (Bidepharm, Shanghai, China), then diluted in 8.0 mL 30% (v/v) PEG400/saline (Bidepharm), ultrasonic mixing. 8 mg/mL compound 42 sodium salt solution was obtained. Fluoxetine, pregabalin and CaCC inh -A01 were also prepared using that same method.

Formalin test
The formalin experiment with reference to the experimental method was reported by Okuda et al. 42 12 SD rats (male, 220e300 g) in each group. The control group and the vehicle group were administrated with normal saline. After 30 min, 5% (v/v) formalin 50 mL was injected into the rat's left-hand foot in the vehicle group and compound group, and 50 mL normal saline was injected in the control group. Then, the number of pain responses (foot lifting) was immediately observed and recorded. 0e10 min after the formalin injection was defined as Phase 1, and 10e60 min was defined as Phase 2. SPSS statistical software was used for data analysis.

Chronic constriction injury model
8 SD rats (male, 6e8 weeks, 180e200 g) in each group. The rat's right hind sciatic nerve was ligation to make CCI model according to the method reported by Chaplan et al. [43][44][45] Paw withdrawal threshold (PWT) was measured 14 days after surgery to evaluate the success of the CCI model (at least 25% lower than before the operation). The PWT was measured 1 h after compound administration. The electronic ciliary instrument used Electronic Von Frey 2390 (IITC Life Science Inc., Woodland Hills, CA, USA). The rate of pain threshold increase (PTI) was calculated as comparable data represent the analgesic effect of different compounds according to Eq. (4): PWT 0 h means pain withdraw threshold before compound administration; PWT 1 h means pain withdraws threshold 1 h after compound administration.

Hot plate test
The hot plate test method is based on that previously reported by Masocha 46 . 10 ICR mice (female, 6e8 weeks, 18e24 g) in each group. The hot plate temperature was 60 C; The Latency record as the time between mice contacted with a hot plate to pain response appeared (foot lifting and jumping) before (0 h) and 3 h after compound administration. The max positive effect (MPE, %) which represents the compound analgesic capability was calculated as Eq. (5): Lat means latency, statistical significance was determined by a paired t-test between 0 and 3 h in each mice.

Writhing test
10 ICR mice (female and male mice (1:1), 6e8 weeks, 18e24 g) each group. The compounds were administered by i.g. administration, and 1 h later, 0.6% (v/v) acetic acid solution was injected by i.p. administration. The number of body writhing was observed and recorded in 15 min. The inhibition rate of compounds was calculated as Eq. (6): N vehicle means the number of writhing in the vehicle group; N comp means the number of writhing in compound treated group; oneway ANOVA was used for intergroup comparison and a paired t-test was used for intra-group comparison.