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A time‐resolved fluorescence resonance energy transfer‐based screen for identification of a novel claudin‐4 binder that modulates the epithelial tight junction barrier
Author(s) -
Kondoh Masuo,
Watari Akihiro,
Yagi Kiyohito
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.00086
Subject(s) - claudin , paracellular transport , tight junction , chemistry , förster resonance energy transfer , biophysics , thiostrepton , microbiology and biotechnology , biochemistry , biology , fluorescence , permeability (electromagnetism) , ribosome , gene , physics , quantum mechanics , membrane , rna
Claudins, a 27‐member family of proteins with four transmembrane domains each, play a pivotal role in maintaining tight junction (TJ) seals in epithelial and endothelial tissue. The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) binds to claudin‐4, which is a key functional and structural component of the TJ in mucosal epithelial cells. Upon binding, C‐CPE reversibly modulates TJ seals, yielding an enhancement of the paracellular transport of solutes. However, the use of C‐CPE as an absorption enhancer is limited by the molecule’s immunogenicity and manufacturing cost. In the present study, we developed a high‐throughput screening system using Time‐Resolved Fluorescence Resonance Energy Transfer (TR‐FRET) to identify small‐molecule compounds that bind to claudin‐4. Using this system, we performed a claudin‐4 binding screen of a library containing 2642 biologically active small molecules, and identified thiostrepton as a novel claudin‐4 binder. Thiostrepton decreased the transepithelial electrical resistance and increased flux of 4‐kDa FITC‐dextran in Caco‐2 cell monolayers without cell toxicity. Immunoblotting analysis revealed that thiostrepton changed the level of claudin‐4 in the Triton‐X soluble fraction, which contains TJ components, without altering the localization of claudin‐4. Furthermore, treatment of isolated rat jejunum with thiostrepton increased absorption of 4‐kDa FITC‐dextran. The TR‐FRET‐based screening system described here is expected to be of use in identifying compounds that modulate the permeability of the intestinal epithelial cell barrier; these compounds might serve as novel absorption enhancers in the intestine. Support or Funding Information This study was supported in part by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19H04468, 18K19400); a grant for Research on Development of New Drugs from the Japan Agency for Medical Research and Development (AMED); and grants from the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) of AMED (JP19am0101077, JP19am0101084, JP19am0101090).