A strategy to identify stable membrane-permeant peptide inhibitors of myosin light chain kinase.

PURPOSE: A peptide inhibitor of myosin light chain kinase (MLCK), termed membrane permeant inhibitor of myosin light chain kinase (PIK), has previously been demonstrated to correct paracellular barrier defects associated with in vitro cell models of infectious and inflammatory intestinal disease. The current study describes a strategy to identify stable analogues of PIK required for future in vivo studies that has resulted in the identification of two promising candidates.
METHODS: Because PIK functions at an intracellular site of epithelial cells and is envisaged to be administered orally, hydrolysis patterns were determined for PIK in both extracts of homogenized Caco-2 (a human intestinal epithelial cell line) and in luminal secretions isolated from rat intestine. Based on these hydrolysis patterns, four peptides Ac-RKKYKYRRK-NH(2) (acetylated PIK), rkkykyrrk-NH(2) (D PIK), krrykykkr-NH(2) (Dreverse PIK), and RKKykyRRK-NH(2) (Dpalindrome PIK) were synthesised. Studies were carried out to determine the stability, activity, and selectivity of these PIK analogues.
RESULTS: D PIK and Dreverse PIK had much longer half-lives of 3.6 and 13.4 h, respectively, compared to PIK, acetylated (Ac)-PIK, or Dpalindrome PIK. All PIK analogues inhibited MLCK potently, although D PIK was a slightly better inhibitor than the other analogues. Similarly, all PIK analogues enhanced paracellular barrier function in Caco-2 monolayers studied in vitro. No appreciable inhibition of cAMP-dependent protein kinase (PKA) or calcium/calmodulin-dependent protein kinase II (CaMPKII) was detected with any of the analogues.
CONCLUSIONS: PIK is quickly degraded within two enzyme-containing preparations that represent different aspects of the intestinal environment. The PIK analogues D PIK and Dreverse PIK demonstrated extended half-lives in these enzyme preparations while retaining the biological activity and specificity of the parent PIK peptide.