Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicities. Here, we show that a unique domain within the MLCK splice-variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identified a domain-binding small molecule (Divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, TNF-induced MLCK1 recruitment as well as downstream MLC 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.