Discovery of (2-aminophenyl)methanol as a new molecular chaperone that rescues the localization of P123S mutant pendrin stably expressed in HEK293 cells.
Pendred syndrome is the most common form of syndromic deafness. It is associated with a mutation in the SLC26A4 gene that encodes pendrin, which is thought to maintain the ion concentration of endolymph in the inner ear most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene are responsible for sensorineural hearing loss. In this study, we established a stable HEK293 cell line expressing P123S mutant pendrin and developed screening methods for compounds that show pharmacological chaperone activity by image analysis using CellInsight™. Morphological analysis of stained cells in each well of 96-well plates yielded six compounds in the compound library. Furthermore, fluorescence intensity analysis of the intracellular localization of P123S mutant pendrin in HEK293 cells using FLUOVIEW™ and cytotoxicity experiments revealed that (2-aminophenyl)methanol 8 is the most promising 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 0.1mM, respectively, and a sustained effect was observed 12h 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 approximately 1 at 10mM concentration and a sustained effect was not observed even at 6h, (2-aminophenyl)methanol 8 was 100 times more potent and exhibited a longer sustained effect than salicylate. These findings suggest that (2-aminophenyl)methanol 8 is an attractive candidate for therapeutic agent for Pendred syndrome patients.
Department of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan.
Wataru
W
Nabeyama
Laboratory of Medical Science, Course for School Nurse Teacher, Faculty of Education, Ibaraki University, Mito 310-8512, Japan.
Kenji
K
Ishihara
Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Hyun Seung
HS
Ban
Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan; Department of Intelligent Information Systems, Tohoku Bunka Gakuen University, Sendai 981-8511, Japan.
Hiroshi
H
Wada
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan. Electronic address: hiro@res.titech.ac.jp.
Hiroyuki
H
Nakamura
D019943:Amino Acid Substitution; D001592:Benzyl Alcohols / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D002462:Cell Membrane / Q000187:drug effects; D006044:Goiter, Nodular / Q000188:drug therapy; D057809:HEK293 Cells; D006319:Hearing Loss, Sensorineural / Q000188:drug therapy; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D012459:Salicylates / Q000494:pharmacology; D000076988:Sulfate Transporters
D001592:Benzyl Alcohols; D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D012459:Salicylates; D000076988:Sulfate Transporters; C072332:2-aminobenzyl alcohol
Hearing loss; Localization; Misfolding; Molecular chaperone; Morphological analysis; P123S mutant; Pendrin