Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.

Asker Y Khapchaev, Olga A Kazakova, Mikhail V Samsonov, Maria V Sidorova, Valery N Bushuev, Elena L Vilitkevich, Andrey A Az'muko, Alexander S Molokoedov, Zhanna D Bespalova, Vladimir P Shirinsky

Journal of peptide science : an official publication of the European Peptide Society 2016 Nov

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Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Citation

Asker Y Khapchaev, Olga A Kazakova, Mikhail V Samsonov, Maria V Sidorova, Valery N Bushuev, Elena L Vilitkevich, Andrey A Az'muko, Alexander S Molokoedov, Zhanna D Bespalova, Vladimir P Shirinsky. Design of peptidase-resistant peptide inhibitors of myosin light chain kinase. Journal of peptide science : an official publication of the European Peptide Society. 2016 Nov;22(11-12):673-681