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

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.

Effects of structural analogues of apelin-12 in acute myocardial infarction in rats.

OBJECTIVE: To examine cardioprotective effects of Ρ-terminal fragment of adipokine apelin-12 (A12), its novel structural analogue [MeArg(1), NLe(10)]-A12 (I), and [d-Ala(12)]-A12 (II), a putative antagonist of APJ receptor, employing in vivo model of ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Peptides were synthesized by the automatic solid phase method using Fmoc technology. Anesthetized open-chest male Wistar rats were subjected to left anterior descending (LAD) coronary artery occlusion and coronary reperfusion. Hemodynamic variables and electrocardiogram (ECG) were monitored throughout the experiment. Myocardial injury was assessed by infarct size (IS), activity of necrosis markers in plasma, and metabolic state of the area at risk (AAR). RESULTS: Intravenous injection of A12, I, or II at the onset of reperfusion led to a transient reduction of the mean arterial pressure. A12 or I administration decreased the percent ratio of IS/AAR by 40% and 30%, respectively, compared with control animals which received saline. Both peptides improved preservation of high-energy phosphates, reduced lactate accumulation in the AAR, and lowered CK-MB and LDH activities in plasma at the end of reperfusion compared with these indices in control. Treatment with II did not significantly affect either the IS/AAR, % ratio, or activities of both markers of necrosis compared with control. The overall metabolic protection of the AAR in the treated groups increased in the following rank: II < A12 < I. CONCLUSIONS: The structural analogue of apelin-12 [MeArg(1), NLe(10)]-A12 may be a promising basis to create a new drug for the treatment of acute coronary syndrome.