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

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule's immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.

Peptides as tight junction modulators.

The first step in drug absorption is the passage of drug molecules across epithelial cell sheets. Epithelial cell sheets are pivotal for the maintenance of homeostasis in the body by acting as a biological barrier that separates the inside of the body from the outside environment. Intercellular space between the adjacent epithelial cells is tightly sealed by tight junctions (TJs), which prevent solutes from freely moving across the epithelial cell sheets. Modulation of the TJ barrier has been a potent strategy for drug absorption. Absorption enhancers have been investigated since the 1980s, and sodium caprate is clinically used as an absorption enhancer. However, the biochemical constituents and structures of TJs were not elucidated until 1993. Occludin, a tetra-transmembrane protein, was identified to be a structural component of TJs in 1993. Claudin, another tetra-transmembrane protein, was identified as a structural and functional component of TJs in 1998. Modulation of occludin- or claudin-barrier is novel methods to enhance drug absorption. Recently, synthetic TJ-binding peptides, a kinase of claudin and peptide fragments of toxins have been developed. In the present review, we summarize the recent progress in TJ-modulating peptides and discuss their potencies.