Potential for Tight Junction Protein-Directed Drug Development Using Claudin Binders and Angubindin-1.

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood-brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development.

LSR antibody promotes apoptosis and disrupts epithelial barriers via signal pathways in endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 µg/ml LSR-N-ab or 2.5 µg/ml angubindin-1 with or without protein tyrosine kinase 2ß inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 µM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption.

A Method to Prepare a Bioprobe for Regulatory Science of the Drug Delivery System to the Brain: An Angulin Binder to Modulate Tricellular Tight Junction-Seal.

Epithelium acts as a barrier separating the interior and exterior of the body, and the epithelial and endothelial cells form tight junctions (TJs) by sealing the paracellular space. The blood-brain barrier (BBB) endothelial cells have well-developed TJs and express specific polarized transport systems to tightly control paracellular movements of solutes, ions, and water. Thus, more than 98% of small-molecular-weight drugs cannot pass the BBB. The tricellular TJ (tTJ) is a structure at contacts of three cells. Angulin-1, also known as lipolysis-stimulated lipoprotein receptor (LSR), is one of angulin family and is abundantly expressed in brain endothelial cells, which plays an important role in barrier function of the BBB. The C-terminal domain of a receptor-binding component of Clostridium perfringens iota-toxin (Ib421-664), also named as angubindin-1, binds to its receptors angulin-1 and angulin-3. This angubindin-1 modulates the tTJ barrier and is able to deliver a 16-mer gapmer antisense oligonucleotide (5.3 kDa) without adverse effects. Thus, angulin binders, such as angubindin-1, are useful tools for studying the safety assessment of tTJ-targeted drug delivery and BBB permeability modulation. Here, we provide a protocol for the expression and purification of recombinant angubindin-1 protein as angulin binders, an analysis method for angubindin-1 binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.

Epithelial barrier dysfunction and cell migration induction via JNK/cofilin/actin by angubindin-1.

Angulin-1/LSR is a tricellular tight junction molecule, that plays an important role in maintaining the epithelial and endothelial barriers. The actin cytoskeleton at tricellular contacts also contributes to the maintenance of the epithelial barrier. Loss of angulin-1/LSR enhances the migration of various cancer cells. Angubindin-1 is a novel binder to angulin-1/LSR and angulin-3. It is a peptide generated from the angulin-1 binding site of Clostridium perfringens iota toxin, which affects the actin cytoskeleton and decreases the epithelial and endothelial barrier functions. However, its regulatory mechanisms are not well understood. To investigate the regulatory mechanisms of the epithelial barrier dysfunction and cell migration induction by angubindin-1, we used human endometrial cancer cell line Sawano, which has high LSR expression and the epithelial barrier function. Angubindin-1 decreased LSR expression and the epithelial barrier function and increased cell migration. It inhibited the recovery of the epithelial barrier function in a Ca-switch model. At tricellular contacts, sinking of the membrane and an increase of actin fibers near the junctions were caused by angubindin-1. It dynamically changed F-actin from lines to dot-like structures at tricellular contacts. Angubindin-1 transiently increased the phosphorylation of cofilin and JNK, which are involved in the regulation of the intracellular actin cytoskeleton. Furthermore, knockdown of JNK and the JNK inhibitor SP600125 prevented the decrease of the epithelial barrier function and the increase of cell migration induced by angubindin-1. These findings suggest that angubindin-1 might reversibly regulate the epithelial barrier and cell migration at tricellular contacts via JNK/cofilin/actin cytoskeleton dynamics.

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

The blood-brain barrier (BBB), which is composed of endothelial cells, pericytes, astrocytes, and neurons, separates the brain extracellular fluid from the circulating blood, and maintains the homeostasis of the central nervous system (CNS). The BBB endothelial cells have well-developed tight junctions (TJs) and express specific polarized transport systems to tightly control the paracellular movements of solutes, ions, and water. There are two types of TJs: bicellular TJs (bTJs), which is a structure at the contact of two cells, and tricellular TJs (tTJs), which is a structure at the contact of three cells. Claudin-5 and angulin-1 are important components of bTJs and tTJs in the brain, respectively. Here, we review TJ-modulating bioprobes that enable drug delivery to the brain across the BBB, focusing on claudin-5 and angulin-1.

Angubindin-1 opens the blood-brain barrier in vivo for delivery of antisense oligonucleotide to the central nervous system.

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.