Regulation of paracellular permeability is essential for epithelial function, while dysregulated permeability is common in disease. Studies of the epithelial tight junction complex have revealed diverse mediators of paracellular permeability, including myosin light chain kinase (MLCK), which induces contraction of the perijunctional actomyosin ring (PAMR), through myosin II regulatory light chain (MLC) phosphorylation. Although MLCK would seem like good a good candidate as a therapeutic target as a regulator of tight junction permeability, unrestricted enzymatic inhibition of MLCK has unacceptable toxicity.
We found that the splice variant of MLCK (MLCK1) directs PAMR recruitment. Moreover, we characterized a unique domain within MLCK1, and identified a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions