title: Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.
abstract: 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-(NαMe)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 by1H-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-(NαMe)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.
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Asker Y
authors.initials: AY
authors.lastname: Khapchaev
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Kazakova
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Samsonov
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Sidorova
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Valery N
authors.initials: VN
authors.lastname: Bushuev
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Elena L
authors.initials: EL
authors.lastname: Vilitkevich
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Az'muko
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Molokoedov
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Zhanna D
authors.initials: ZD
authors.lastname: Bespalova
authors.affiliation: Russian Cardiology Research and Production Center, 3rd Cherepkovskaya St., 15a, Moscow, 121552, Russia.
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Shirinsky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D001923:Brain Chemistry; D002417:Cattle; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000097:blood / Q000138:chemical synthesis* / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D005895:Gizzard, Avian / Q000737:chemistry; D006207:Half-Life; D006801:Humans; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000097:blood / Q000138:chemical synthesis* / Q000494:pharmacology; D055550:Protein Stability; D059748:Proteolysis; D060327:Solid-Phase Synthesis Techniques / Q000379:methods; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014422:Turkeys
chemical_list: D030161:Avian Proteins; D057846:Cell-Penetrating Peptides; D047428:Protein Kinase Inhibitors; D009219:Myosin-Light-Chain Kinase; D013917:Thrombin
keywords: degradation in plasma; endothelial permeability; myosin light chain kinase; myosin regulatory light chains; nuclear magnetic resonance; peptide inhibitor; phosphorylation; solid phase synthesis