Abstract
GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10–1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.
Highlights
Failure of mucosal healing is central to diseases as diverse as inflammatory bowel disease (IBD), peptic ulcer, and necrotizing enterocolitis (NEC)
Our observations presented here suggest that such drug-like small molecules can promote intestinal epithelial restitution in vitro and in mucosal healing in mice, at least in part by activating Focal adhesion kinase (FAK)
Because we were interested in the potential effects of these compounds on epithelial sheet migration, we evaluated the effect of ZINC40099027 in migrating Caco-2 cells, using a model of sparse seeding to create small islands of migrating cells as previously described[5]
Summary
Failure of mucosal healing is central to diseases as diverse as inflammatory bowel disease (IBD), peptic ulcer, and necrotizing enterocolitis (NEC). GI mucosal healing represents an equilibrium between injurious agents and the migration and proliferation of epithelial cells at the wound edge. Activated FAK is decreased in migrating intestinal epithelial cells in vitro[5] and at the edge of human mucosal ulcers[6], making FAK an attractive target to promote mucosal healing. (b) Representative blots and Tyr-397/FAK fold change treated with ZINC40099027 in confluent adherent static Caco-2 cells. (c) Representative blots and Tyr-397/ FAK fold change in migrating cells at various time points after treatment with 10 nM ZINC40099027(n = 7, *p < 0.05). ZINC4009027, would promote intestinal epithelial monolayer wound closure via sheet migration in vitro, the extent to which the effects are due to FAK activation and to the stimulation, and the potential efficacy of one of these molecules in vivo
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