Identification of claudin-4 binder that attenuates tight junction barrier function by TR-FRET-based screening assay

We developed a high-throughput screening system based on the TimeResolved Fluorescence Resonance Energy Transfer method to identify claudin-4 binders in a library collection of 32,560 compounds

Akihiro Watari; Miki Kodaka; Koji Matsuhisa; Yuta Sakamoto; Kota Hisaie; Norihito Kawashita; Tatsuya Takagi; Yoshiaki Yamagishi; Hidehiko Suzuki; Hirofumi Tsujino; Kiyohito Yagi; Masuo Kondoh

2017

Scholarcy highlights

  • Claudins are key functional and structural components of tight junctions in epithelial cell sheets
  • C-terminal fragment of Clostridium perfringens enterotoxin is highly active in lowering TJ barrier function, suggesting that the region of interaction between C-CPE and claudin-4 is pivotal in the disruption of claudin-4-mediated TJ barrier formation; and interactions between other claudin-4 binders and this region have the potential to disrupt TJ barriers and enhance absorption via the paracellular route
  • Adding C-CPE or the antibody recognising the extracellular domain of claudin-4 considerably reduced the FRET signal, whereas adding C-CPE Y306A/L315A or the antibody recognising the C-terminal of claudin-4 did not affect the signals, indicating that this assay can be used to identify binders that bind to the extracellular domain of claudin-4
  • We constructed a claudin-4 binder screening system based on TimeResolved Fluorescence Resonance Energy Transfer, which detects the interaction between claudin-4 and C-CPE
  • We surmise that our novel screening system will simplify the identification of compounds binding to claudin-4 but will restrict the outcome to compounds with TJ barrier-regulating activity, given that the compounds obtained bind substantially to the claudin-4 region that interacts with C-CPE
  • Thiostrepton treatment increased the 1% Triton-X-soluble protein and mRNA levels, and the 1% Triton-X-insoluble protein levels, of claudin-4
  • We showed that TR-FRET analysis distinguished between an antibody that recognises the intracellular domain of claudin-4, which cannot regulate TJ barriers, and one that recognises the extracellular domain of claudin-4, which attenuates TJ-barrier function
  • For analysis of thiostrepton-induced cytotoxicity on Caco-2 cells, a lactate dehydrogenase release assay was performed according to the manufacturer’s instructions

Need more features? Save interactive summary cards to your Scholarcy Library.