Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Discovery of (2-aminophenyl)methanol as a new molecular chaperonethat rescues the localization of P123S mutant pendrin stably expressedin HEK293 cells

http://dx.doi.org/10.1016/j.bmc.2017.03.0240968-0896/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: hiro@res.titech.ac.jp (H. Nakamura).

Wataru Nabeyama a, Kenji Ishihara b, Hyun Seung Ban c, Hiroshi Wada d,e, Hiroyuki Nakamura f,⇑aDepartment of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japanb Laboratory of Medical Science, Course for School Nurse Teacher, Faculty of Education, Ibaraki University, Mito 310-8512, JapancMetabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of KoreadDepartment of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579, JapaneDepartment of Intelligent Information Systems, Tohoku Bunka Gakuen University, Sendai 981-8511, Japanf Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan

a r t i c l e i n f o

Article history:Received 21 February 2017Revised 11 March 2017Accepted 11 March 2017Available online 14 March 2017

Keywords:Hearing lossPendrinP123S mutantMisfoldingMolecular chaperoneLocalizationMorphological analysis

a b s t r a c t

Pendred syndrome is the most common form of syndromic deafness. It is associated with a mutation inthe SLC26A4 gene that encodes pendrin, which is thought to maintain the ion concentration of endolymphin the inner ear most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4gene are responsible for sensorineural hearing loss. In this study, we established a stable HEK293 cell lineexpressing P123S mutant pendrin and developed screening methods for compounds that show pharma-cological chaperone activity by image analysis using CellInsightTM. Morphological analysis of stained cellsin each well of 96-well plates yielded six compounds in the compound library. Furthermore, fluorescenceintensity analysis of the intracellular localization of P123S mutant pendrin in HEK293 cells usingFLUOVIEWTM and cytotoxicity experiments revealed that (2-aminophenyl)methanol 8 is the most promis-ing molecular chaperone to rescue P123S mutant pendrin: the plasma membrane (M)/cytoplasm (C)ratios are 1.5 and 0.9 at the concentrations of 0.3 and 0.1 mM, respectively, and a sustained effect wasobserved 12 h after removal of the compound from the cell medium. Because the M/C ratio of salicylate,which was previously discovered as a molecular chaperone of P123S mutant pendrin, was approximately1 at 10 mM concentration and a sustained effect was not observed even at 6 h, (2-aminophenyl)methanol8 was 100 times more potent and exhibited a longer sustained effect than salicylate. These findingssuggest that (2-aminophenyl)methanol 8 is an attractive candidate for therapeutic agent for Pendredsyndrome patients.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Pendred syndrome is the most common form of syndromicdeafness and considered to account for up to 10% of all cases ofsyndromic hearing loss.1 Pendred syndrome is associated with amutation in the SLC26A4 gene that encodes pendrin.2–4 Pendrin isan 85.7 kDa membrane protein and a member of the anion trans-porter family SLC26 that maintains the ion concentration of endo-lymph in the inner ear, most likely by acting as a chloride/bicarbonate transporter.5–9 More than 150 mutations have beenidentified in the SLC26A4 gene of humans and these mutations

are responsible for Pendred syndrome and non-syndromic hearingloss with an enlarged vestibular aqueduct (NSEVA).10,3,11

Previously, we analyzed the cellular localization and the aniontransporter activity of 10 missense pendrin mutations found inJapanese patients, including p.P123S (Pendred syndrome),p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syn-drome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA),p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA), andp.H723R (Pendred syndrome/NSEVA),12 using HEK293 cells trans-fected with each mutant gene.13 We found that p.K369E andp.C565Y, as well as wild-type pendrin, were transported to theplasma membrane and the eight other mutants were retained inthe cytoplasm. Interestingly, four of the eight pendrin mutants(p.P123S, p.M147V, p.S657Y, and p.H723R) were transported tothe plasma membrane and the anion-exchange activity was

2602 W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608

restored in the presence of 10 mM of salicylate, revealing that sal-icylate acts as a molecular chaperone14,15 to rescue these pendrinmutants.13 A molecular chaperone corrects these misfolding pen-drin mutants in folding and restores their function. It is thereforenecessary to find compounds that exhibit the pharmacologicalchaperone activity for the treatment of patients with syndromichearing loss.16,17 In this study, we established a screening methodfor compounds that show P123S mutant pendrin chaperone activ-ity using P123S mutant pendrin stably expressed in human embry-onic kidney (HEK) 293 cells, in hopes of finding morepharmacologically active compounds to rescue mutant pendrin.

2. Materials and methods

2.1. Expression vectors

Expression vectors of human pendrin wild-type (WT; AccessionNo. NM_000441.1.) and P123S mutant were constructed in a previ-ous study.13 In brief, cDNA of wild type or P123S mutant fused with3�FLAG tag at its C-terminus was cloned into the pcDNA3.1 mam-malian expression plasmid vector. The expression plasmids wereamplified in E. coli (JM109) and purified from the cells using a Plas-mid Mini Kit (QIAGEN, Germantown, MD).

2.2. Cell culture and construction of stably expressed cell lines of WTand P123S pendrins in HEK293 cells

HEK293 cells were maintained in RPMI-1640 medium (Sigma,St. Louis, MO) supplemented with 10% fetal bovine serum (FBS)at 37 �C with 5% CO2. After 12 h, the cells were transfected withWT and P123S plasmid DNAs by using FuGENE� HD transfectionreagent (Promega, Madison, WI) according to the manufacturer’sprotocol. These two types of cells were seeded in 10% FBS-RPMI-1640 medium containing 500 lg/ml G-418 Sulfate (Calbiochem�,La Jolla, CA) for 10 days and neomycin-resistant cells wereobtained. The neomycin-resistant cells were cloned with the limit-ing dilution-culture method in medium containing 500 lg/ml G-418 Sulfate (Calbiochem�) on duplicate 96-well culture plates.The cells on one of two plates were subjected to immunofluores-cence staining in the following way: After the cells were washedwith PBS three times, they were fixed in 4% paraformaldehydefor 5 min. Then, the cells were blocked with 1% BSA-PBS for 1 h.Mouse anti-FLAG� M2-FITC antibody (Sigma) and rhodamine-con-jugated phalloidin (Cytoskeleton, Inc., Denver, CO) were added to

OH

Salicylate

OH

Salicyl alcohol

NH2

1

CO2HOH

CO2H

OH

5

CO2HO2N

6

CO2H

HO

HO

SH

7

CO

OH

11

CO2H

O2N OH

12

CO2HH2N

13

HO

16

OH

NH2 17

OH

H2N

1

O

Fig. 1. Chemical structures of salicylate and its deriva

the cells for pendrin imaging and cell skeleton imaging, respec-tively. The cells were incubated at room temperature for 1 h andwashed with PBS three times. Immunofluorescence images wereobtained with a confocal laser scanning microscope (FV 500, Olym-pus Co., Tokyo, Japan). Then, the wells containing pendrin-positivecells were selected in the other 96-well plate and the cells obtainedin each well were evaluated for salicylate-induced molecular chap-erone activity. Cells positive for salicylate-induced molecular chap-erone activity were cloned repeatedly in the same manner andused in further experiments.

2.3. Immunofluorescence microscopy

WT cells and P123S cells were seeded in a 96-well culture plateor a 3.5 cm culture dish containing 10% FBS-RPMI-1640 medium.P123S cells were incubated in the presence of vehicle (0.1% DMSO),salicylate, and salicylate derivatives (Fig. 1) at the specified con-centration of each compound for 12 h. WT cells were incubatedin the presence of vehicle (0.1% DMSO) for 12 h as positive control.Salicylate and salicylate derivatives were purchased from Sigma-Aldrich Co. LLC. (St. Lois, MU).

The cells were subjected to immunofluorescence staining in thefollowing way: After the cells were washed with PBS three times,they were fixed in 4% paraformaldehyde for 5 min. Then, the cellswere blocked with 1% BSA-PBS for 1 h. Mouse anti-FLAG� primaryantibody (Sigma) was added to the cells and the cells were incu-bated at room temperature for 1 h and washed with PBS threetimes. The cells were incubated in the presence of anti-mouse sec-ondary antibody conjugated with Alexa Fluor� 546 (Invitrogen,Carlsbad, CA) and DAPI (Wako Pure Chemicals Ltd. Osaka, Japan)at room temperature for 1 h and then washed three times withPBS. Fluorescence images of the cells were obtained with the con-focal laser scanning microscope and analyzed by FLUOVIEWTM

(Olympus, Co.).

2.4. Localization of P123S mutant pendrin in HEK293 cells

Immunofluorescent cells on the 96-well culture plate were ana-lyzed by CellInsightTM (Thermo Fisher Scientific Inc., Waltham, MA).The cells in each well were divided into 100 fields to detect approx-imately 100 cells in one field. Cell images were analyzed by usingprotocols Ch 1–4 as shown in Fig. 2. Protocol Ch 1 was pro-grammed to recognize the plasma membrane (the outline of cells),whereas protocol Ch 2 was programmed to detect the nucleusstained by DAPI. Protocol Ch 3 was programmed to measure the

OH

2

CO2H

OHOH

3

CO2HOH

OAc

4

CO2H

2H

8

NH2

OH

OH

OH

O

O

9

OH

ONa

O

10

OH

CO2H

OH

14

B(OH)2

OH

15

CO2HH3C

8

H

OH

19

NH2

OH OH

CO2H

20

tives. All compounds are commercially available.

(A)

Ch 3 = 5 pixels

7 pixels

Ch1

Ch 2

Nc

Cytoplasm

Ch 4 = 2 pixels Plasma membrane

)D()C(

Fig. 2. Morphological analysis of immunofluorescent cells in each well by CellInsightTM using image analysis protocols Ch 1–4: (A) plasma membrane (the outline of cells) (Ch1), nucleus stained by DAPI (Ch 2), cytoplasm from 2 to 7 pixels (the field of 5 pixels) within the outline of cells (Ch 3), and plasma membrane for the detection of translocatedpendrins (Ch 4). (B) Morphological analysis of immunofluorescent cells incubated with and without salicylate (10 lM) on the 96-well culture plate measured by CellInsightTM

using image analysis protocols Ch 1–4. Bar indicates 10 lm. (C) Salicylate-induced fluorescence intensities in plasma membrane using protocol Ch 4. (D) Salicylate-inducedfluorescence intensities in cytoplasm using protocol Ch 3.

W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608 2603

fluorescence intensity of the cytoplasm from 2 to 7 pixels (the fieldof 5 pixels) within the plasma membrane of cells recognized byprotocol Ch 1 for the detection of pendrins expressed in the cyto-plasm. Protocol Ch 4 was programmed to measure the fluorescenceintensity of the plasma membrane for the detection of translocatedpendrins.

2.5. Cytotoxicity of compounds toward P123S mutant HEK293 cells

P123S mutant HEK293 cells were seeded in a 96-well cultureplate at 5 � 104 cells/well and incubated at 37 �C for 12 h. Then,the cells were further incubated in RPMI-1640 medium containingcompounds at various concentrations for 72 h. Cell viability was

2604 W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608

determined by the MTT assay. The concentration required toreduce cell viability by 50% (IC50) was determined from thesemilogarithmic dose-response plots. Triplicate experiments werecarried out.

2.6. Sustainability evaluation of chaperone activity of compoundstoward P123S mutant pendrin in HEK293 cells

The cells were seeded in a 96-well culture plate (5 � 104 cells/well) and incubated in the presence of vehicle (0.1% DMSO), salicy-late, and salicylate derivatives for 12 h. The cells were washed withPBS and then further incubated for 0, 6, and 12 h. Sustainabilityevaluation was carried out by measuring the time-dependent dis-tribution of P123S mutant pendrin from the plasma membrane inHEK293 cells. Each sample was stained with anti-FLAG� primaryantibody and DAPI- and immunofluorescence-stained cells were

Fig. 3. Screening for salicylate and its derivatives. WT cells and P123S cells were incubatfor 12 h. The cells were subjected to immunofluorescence staining and morphological animage analysis protocol Ch 3. In the case of compound 8, the experiment was carried o

analyzed in the same manner as that described in Section 2.3. Trip-licate experiments were carried out.

3. Results and discussion

3.1. Localization analysis of P123S mutant pendrin in HEK293 cells

The localization of P123S mutant pendrin in HEK293 cells wasanalyzed by CellInsightTM. We first tried to determine the localiza-tion of P123S mutant pendrin in the plasma membrane directlyusing protocol Ch 4, which was programmed to detect translocatedpendrins. As shown in Fig. 2(C), the dose-dependent increase of thefluorescence intensities of P123S mutant pendrin induced by sali-cylate was not observed as expected, although P123S mutant pen-drin was localized in the plasma membrane of HEK293 cellsinduced by salicylate (see Fig. 4). Therefore, we analyzed the

ed in the presence of vehicle (0.1% DMSO) or compound at 1–30 mM concentrationsalysis of immunofluorescent cells in each well was carried out by CellInsightTM usingut at 0.03–1 mM concentrations.

Fig. 4. Effects of salicylate and its derivatives on the intracellular localization of P123S mutant pendrin in HEK293 cells. The cells were incubated in the presence ofcompounds at the following concentrations (0.1% DMSO (None), salicylate, compounds 5 and 15: 1–10 mM; compound 8: 0.03–0.3 mM) for 12 h. The intracellularlocalization of pendrin was analyzed by immunofluorescence staining with anti-FLAG� antibody and DAPI. Fluorescence intensity analysis of the intracellular localization ofP123S mutant pendrin in HEK293 cells was also carried out by FLUOVIEW (Olympus). The intensities on the horizontal lines in the cell images are plotted in the graphs on theright to indicate the fluorescence intensity of P123S mutant pendrin in plasma membrane and cytoplasm of HEK293 cells. ‘‘Wt” means stably expressed cell lines of wild-typependrin in HEK293. Bar indicates 5 lm.

W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608 2605

fluorescence intensities of P123S mutant pendrin in the cytoplasmof HEK293 cells using protocol Ch 3. As shown in Fig. 2(D), a dose-dependent decrease of the fluorescence intensities of P123Smutant pendrin was observed in the cytoplasm, indicating thatprotocol Ch 3 can be used to detect the pharmacological chaperoneactivity of compounds using stably expressed P123S mutant pen-drin in HEK293 cells.

3.2. Effects of salicylate derivatives on localization of P123S mutantpendrin in HEK293 cells

Because we were able to establish a protocol for the determina-tion of P123S mutant pendrin localization in the plasma membraneof HEK293 cells as discussed in the previous section, we nextexamined a procedure for screening salicylate derivatives withregard to their effects on the localization of P123S mutant pendrin.The results are summarized in Fig. 3. Salicylate, but not salicyl alco-hol, induced the localization of P123S mutant pendrin in plasmamembrane in a concentration-dependent manner. Compounds 1,4, and 7, which have a carboxylic acid moiety in the molecules,did not show the chaperone activity, indicating that a phenolichydroxyl group is essential for the chaperone activity. Compounds

2, 3, 6, 12, and 13 did not show the chaperone activity as well.Compounds 5 and 11, which have an electron-withdrawing groupin the molecules, showed significant chaperone activity, indicatingthat the acidity of benzoylic acids is also important for the chaper-one activity. A benzene ring is also indispensable for the chaperoneactivity (compounds 9 and 10). Interestingly, aniline derivative 8and boronic acid derivative 14 also showed significant chaperoneactivity, suggesting that carboxylic acid can be replaced by amineor boronic acid. 2-(2-Hydroxyethyl)phenol 18, but not 2-(2-hydroxyethyl)aniline 19, possessed significant chaperone activity.Compound 20, which has an ethylene linker between benzene ringand carboxylic acid in salicylate, also exhibited the chaperoneactivity. Among the compounds screened, compounds 5, 8, 11,14, 15, and 18 showed significant chaperone activity to rescueP123S mutant pendrin.

3.3. Effects of salicylate derivatives on cell viability of P123S mutantHEK293 cells

Proliferation and cell viability are also an important considera-tion when examining the chaperone activity of compounds for therescue of P123S mutant pendrin. Therefore, we investigated the

Table 1Cytotoxicity of compounds toward P123S mutant HEK293 cells.

Compound IC50 (mM)a Compound IC50 (mM)

Salicylate >15 (4.2%) 10 >15Salicyl alcohol >15 (25.3%) 11 12.5 ± 2.411 >15 (46.9%) 12 >15 (35.5%)2 >15 (47.4%) 13 >15 (46.8%)3 10.6 ± 2.6 14 >15 (22.3%)4 10.2 ± 0.62 15 9.8 ± 0.485 5.3 ± 0.87 16 >15 (46.7%)6 >15 (37.0%) 17 12.7 ± 0.857 12.7 ± 1.8 18 >15 (38.3%)8 3.6 ± 0.57 19 >15 (35.8%)9 9.7 ± 0.71 20 12.1 ± 0.86

a Percentage inhibition of cell viability at 15 mM is indicated in parenthesis.

2606 W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608

cytotoxicity of compounds toward P123S mutant HEK293 cells. Asshown in Table 1, salicylate exhibited 4.2% inhibition of cell viabil-ity at 15 mM. The other compounds are non-cytotoxic or have lowcytotoxicity similar to salicylate. Compounds 5 and 8 exhibitedcytotoxicity and their IC50 values were 5.3 and 3.6 mM, respec-tively. Although the cell growth inhibitions of compound 8 were43.1 and 20.8% at 1.0 and 0.3 mM, respectively, no inhibitory activ-ity was observed at 0.1 mM concentration.

3.4. Effects of salicylate derivatives on P123S mutant pendrintranslocation in HEK293 cells

We next analyzed the effects of salicylate derivatives, whichwere found to have more potential than salicylate in the screening,on the translocation of P123S mutant pendrin into the plasmamembrane of HEK293 cells. Stable HEK293 cell line expressing

Fig. 5. Fluorescence intensities of plasma membrane (M) and cytoplasm (C) of cells werethe mean fluorescence intensities of 20 regions (0.8 mm � 0.8 mm) in plasma membran

pendrin mutant P123S was incubated with the salicylate deriva-tives in 35 mm dishes for 12 h and the cells were subjected toimmunofluorescence staining for analysis under a fluorescencemicroscope. As compounds 11, 14, and 18 induced the aggregationof cells after incubation for 12 h, the localization of P123S mutantpendrin in HEK293 cells could not be imaged by fluorescencemicroscopy. These results indicated that cell aggregation couldnot be properly detected by CellInsight. Therefore, we analyzedthe effects of compounds 5, 8, and 15 on the intracellular localiza-tion of P123S mutant pendrin in HEK293 cells. Salicylate was usedas positive control. As shown in Fig. 4, P123S mutant pendrin waslocalized in the cytoplasm in the absence of any additives. How-ever, it was translocated into the cell membrane in the presenceof 10 mM salicylate and this localization was similar to that ofwild-type pendrin in HEK293 cells. The translocation of P123Smutant pendrin into the cell membrane was also observed in thepresence of 3 mM compound 5. Surprisingly, compound 8 thathas no carboxylic acid in the molecule showed significant chaper-one activity: the complete translocation of P123S mutant pendrininto the plasma membrane was observed even at the concentrationof 0.1 mM, indicating that compound 8 possesses 100 times higherchaperone activity than salicylate. Compound 15, which has amethyl group in the molecule, showed similar chaperone activityto salicylate.

Fluorescence intensities were analyzed by FLUOVIEW (Olym-pus, Co.) to compare the effect of compounds on the intracellularlocalization of P123S mutant pendrin in HEK293 cells. The analysiswas carried out on the basis of the fluorescence images in Fig. 4and the concentration-dependent fluorescence intensities of theplasma membrane and the cytoplasm of the cells were evaluated,as shown in Fig. 5. These analyses were carried out according toour previously reported protocols. In the case of salicylate, the

evaluated by FLUOVIEW analysis in Fig. 5. M/C ratios were obtained by calculatinge and cytoplasm. Values are means of 6–9 cells.

Fig. 6. Sustainable effects of salicylate and its derivatives 5, 8, and 15 on the intracellular localization of P123S mutant pendrin in HEK293 cells after removal of compoundsfrom the medium. Bar indicates 5 lm.

W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608 2607

fluorescence intensities of the cytoplasm (C) decreased with theincrease of the fluorescence intensities of the plasma membrane(M) in a concentration-dependent manner, and the M/C ratio wasapproximately 1 at 10 mM concentration (Fig. 5(A), line plot). Incontrast, the M/C ratio of wild-type pendrin in HEK293 cells wasapproximately 1.8 (data not shown). Similar to salicylate, salicylatederivatives 5, 8, and 15 also showed a concentration-dependentincrease of the M/C ratios. The M/C ratio of compound 5 is approx-imately 1.3 at 10 mM concentration and this value is higher thanthat of salicylate. However, the cell growth inhibition of compound5 is relatively high (IC50: 5.3 mM) compared to salicylate. There-fore, compound 5 is not suitable as a pharmacological chaperonemolecule because of its cytotoxicity. The M/C ratio of compound15 is approximately 1.7 at 10 mM concentration, and this concen-tration is almost the same as its IC50 (9.8 mM). In contrast, thehighest M/C ratio was obtained for compound 8: it is approxi-mately 1.8 even at 0.3 mM concentration. Furthermore, the M/Cratio of compound 8 is 0.9 at 0.1 mM concentration that is similarto that of salicylate at 10 mM concentration. Because the IC50 ofcompound 8 is 3.6 mM, the effective concentration for the chaper-one activity toward P123S mutant pendrin in HEK293 cells is stillmuch lower than the IC50.

3.5. Sustainability evaluation of chaperone activity of compoundstoward P123S mutant pendrin in HEK293 cells

Finally, we investigated the sustainability of the chaperoneactivity of the compounds toward P123S mutant pendrin in

HEK293 cells. The cells were incubated with the compounds at10 mM (0.1 mM for compound 8) for 12 h and the compoundswere removed from the cell medium. The time-dependent intracel-lular localization of P123S mutant pendrin in HEK293 cells wasmeasured by fluorescence microscopy after incubation in freshmedium. The results are shown in Fig. 6. P123S mutant pendrinwas localized in the cytosol of HEK293 cells, whereas it wastranslocated into the plasma membrane in the presence of10 mM salicylate. However, P123S mutant pendrin was delocalizedin the cytosol 6 h after salicylate was removed from the medium.Interestingly, P123S mutant pendrins treated with compounds 5,8, and 15 were still detected in the plasma membrane 6 h afterthe compounds were removed from the medium. The sustainableeffects were observed even after 12 h in cells treated with com-pounds 5 and 8. Indeed, a large population of P123S mutant pen-drins were still localized near the plasma membrane in cellstreated with compound 8, indicating that compound 8 is the mostsuitable molecular chaperone to rescue P123S mutant pendrinamong the compounds screened in the current study.

4. Conclusion

We established a stable HEK293cell line expressing pendrinmutant P123S. Using the cell line, we developed a screeningmethod for compounds that show P123S mutant pendrin chaper-one activity by image analysis using CellInsightTM. Morphologicalanalysis of stained cells in each well of 96-well plates yielded six

2608 W. Nabeyama et al. / Bioorganic & Medicinal Chemistry 25 (2017) 2601–2608

compounds 5, 8, 11, 14, 15, and 18 in the compound library, asshown in Fig. 1. Fluorescence intensity analysis of the intracellularlocalization of P123S mutant pendrin in HEK293 cells using FLUO-VIEWTM and cytotoxicity experiments revealed that compound 8possessed the most promising molecular chaperone activity to res-cue P123S mutant pendrin: the M/C ratios were 1.5 and 0.9 at 0.3and 0.1 mM concentrations, respectively. As the M/C ratio of salicy-late was 1.0 at 10 mM concentration, compound 8 was able toexhibit chaperone activity toward P123S mutant pendrin at 100times lower concentration than salicylate. Our current establishedhigh-through-putt cell screening technology is based on the mor-phology analysis by fluorescent microscopy. Not only the integra-tion of fluorescence but also translocation of proteins of interestcan analyzed. Indeed, it has been reported that misholdingmutants of membrane proteins such as V2 vasopressin receptor,P-glycoprotein and GnRH receptor can be rescued by small molec-ular compounds as molecular chaperones.18,16 Therefore, the cur-rent technology can be applicable in a wide variety of analysis ofefficiencies of biologically active molecules including small com-pounds toward cell morphology including suppression and/orinduction of gene expression and protein localization in cells. Inaddition, compound 8 showed the largest sustainable effect amongthe compounds screened for chaperone activity. Together, ourresults suggest that compound 8 is an attractive candidate for ther-apeutic agent for Pendred syndrome patients. Further studies ofthe effects of compounds on other pendrin mutations are inprogress.

Acknowledgment

This work was performed under the Cooperative Research Pro-gram ‘‘Network Joint Research Center for Materials and Devices”from Ministry and Education, Culture, Sports, Science and Technol-ogy (MEXT) Japan.

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