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Novel small molecules reverse myosin light chain kinase (MLCK) isoform 1 translocation and barrier dysfunction induced by tumor necrosis factor (TNF)
Author(s) -
Graham W. Vallen,
Marchiando Amanda M.,
Wang Yingmin,
Ostrov David A.,
Turner Jerrold R.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1183.8
Subject(s) - myosin light chain kinase , chemistry , tumor necrosis factor alpha , myosin , barrier function , in vivo , chromosomal translocation , tight junction , microbiology and biotechnology , biochemistry , biology , endocrinology , genetics , gene
MLCK is central to TNF‐induced intestinal epithelial barrier dysfunction in vitro and in vivo . MLCK is expressed as two splice‐variants, MLCK1 and MLCK2, distinguished by a short insertion in MLCK1. MLCK1 knockdown increases epithelial barrier function in vitro . We therefore hypothesized that MLCK1 might specifically regulate the perijunctional actomyosin ring (PAMR) during TNF‐induced barrier dysfunction. TNF‐treated Caco‐2 monolayers demonstrated a progressive increase in PAMR‐associated MLCK1 within 30 min, 35.7±0.1% ( P <0.05), that peaked at 4 hr, 118.8±0.1% ( P <0.001), and correlated directly with TNF‐induced barrier loss (r=0.9). Structural modeling showed that the MLCK1 insertion created a binding pocket. In silico screening identified candidate small molecules targeting this pocket, of which, one molecule, SMKI2, bound the pocket with a predicted ΔG of −92 kcal/mole. 59.9±0.2% ( P <0.001) of MLCK1 was displaced from the PAMR by 250 μM SMKI2. In contrast, enzymatic MLCK inhibition caused no MLCK1 displacement. However, like enzymatic inhibition, SMKI2 corrected TNF‐induced barrier loss in a dose‐dependent fashion. These data provide insight into the mechanism of TNF‐induced MLCK activation and PAMR myosin phosphorylation. Inhibition of MLCK1 targeting may be an important means of correcting barrier dysfunction without the potential toxicities of enzymatic MLCK inhibition. Supported by NIH