Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Department of Stress Protein Processing, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Sci Rep. 2017 Nov 6;7(1):14514. doi: 10.1038/s41598-017-15108-y.
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.
紧密连接（TJ）中的 Claudin 是关键的功能和结构组成部分，上皮细胞片。产气荚膜梭菌肠毒素的 C 端片段（C-CPE）与 Claudin-4 结合，并可逆地调节肠道 TJ 密封，从而增强溶质的旁细胞转运。然而，C-CPE 作为吸收增强剂的使用受到分子免疫原性和制造成本的限制。在这里，我们开发了一种基于时间分辨荧光共振能量转移（TR-FRET）方法的高通量筛选系统，以从 32560 种化合物的文库中鉴定 Claudin-4 结合物。从筛选中鉴定出的硫链丝菌素降低了 Caco-2 细胞单层的跨上皮电阻并增加了 4 kDa 荧光素异硫氰酸酯标记的葡聚糖（FD-4）的通量，这是肠道上皮的模型。硫链丝菌素改变了 TJ 成分的表达，但不改变其定位。硫链丝菌素处理大鼠空肠增加了 FD-4 的吸收而没有组织毒性，表明硫链丝菌素是一种增强肠道通透性的新型 Claudin-4 结合物。因此，该筛选系统可能是一种有用的工具，可用于鉴定 Claudin-4 结合物以增强粘膜中的药物吸收。
