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

Abstract

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.

Single-cell analysis of colonic epithelium reveals unexpected shifts in cellular composition and molecular phenotype in treatment-naïve adult Crohn’s disease

“…This is concordant with our finding of a shift toward heightened goblet maturation in CD compared to NIBD (Figure 2E). Interestingly, levels of CLDN4, previously reported to be highly expressed in EECs (Nagatake et al, 2014)and also studied in the context of colonocyte barrier function (Watari et al, 2017), are significantly elevated in the mature goblet cell cluster while modestly reduced in both CEACAM7+ colonocytes and EECs ( Figure 3F). In fact, in CD, the levels of CLDN4 in mature goblet cells rise to what is observed in CEACAM7+ colonocytes ( Figure 3F), the cells in which CLDN4 is most highly expressed in NIBD.…”

“…Future single-cell studies must expand on this work to include larger cohorts and to assess the impact of age, gender, CD sub-type (Keith et al, 2018;Weiser et al, 2018), disease region (e.g., ileum vs. colon), disease duration, and treatment history on cell composition and molecular phenotype. Longitudinal investigations to uncover changes to the cellular landscape and molecular phenotype over the course of disease progression are also merited.…”

Evaluation of Sucrose Laurate as an Intestinal Permeation Enhancer for Macromolecules: Ex Vivo and In Vivo Studies

“…Even so, it appears that oral relative bioavailability of peptides in humans is limited to ~1–2% with Generation 1 PEs [5], thus their application in once-a-day oral dosing must ideally be matched to stable macromolecules with long half-lives [6,7]. Although it would seem that tight junction-specific agents targeted at enzymatic pathways [8] or claudin-4 [9] might have less capacity for epithelial toxicity than agents that cause perturbation through non-specific actions, second-generation PE molecules for oral delivery remain in preclinical studies likely because of low stability, high cost as well as formulation and manufacturing issues. In addition, Pharma has an understandable aversion to the increased financial and regulatory risk associated with using new chemical entity PEs in oral macromolecule formulations—unless they can provide more than an incremental increase in efficacy.…”

Tight Junction Modulating Bioprobes for Drug Delivery System to the Brain: A Review

“…Furthermore, recently, Watari et al, reported a high-throughput screening system based on the time-resolved fluorescence resonance energy transfer method to identify claudin-4 binders. They identified several claudin-4 binders with epithelial-barrier-disrupting activity, such as thiostrepton, using the developed method [ 113 ]. These strategies are expected to accelerate the development of new claudin binders that modulate the BBB function in the future.…”