Peptidase-resistant peptide inhibitors of myosin light chain kinase

Abstract

Myosin light chain kinase (MLCK) is the key regulator of various forms of cell motility including endothelial and epithelial permeability in particular. One of the potential MLCK inhibitors to be used in humans is a membrane permeable peptide H-RKKYKYRRK-NH2 (L-PIK). In present work we used solid phase peptide synthesis and Fmoc-technology to produce five modifications of L-PIK. Based on (1)H NMR analysis revealed that these peptides demonstrated improved resistance to degradation in blood plasma. One of de novo synthesized peptides, L-[MeArg(1)]PIK inhibited MLCK activity in vitro with the same efficiency as L-PIK whereas other modified peptides showed reduced inhibitory activity. D-amino acid analog of PIK was the least active inhibitor. Thus, we have demonstrated the possibility to produce an effective MLCK peptide inhibitor with increased resistance to biodegradation that is suitable for further pharmacological development.

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

“…The rate of cleavage at Arg 1 decreased about three-fold compared to an unprotected residue and nearly equalized with the rate of peptide hydrolysis at the C-terminus. Overall, the proteolytic stability of PIK4 was similar to that of PIK1 [12]: both peptides were 50% degraded after 2-h incubation in blood plasma. Finally, cyclization of L-PIK was moderately effective in protecting the peptide from plasma peptidases.…”

“…None of the novel PIK versions demonstrated resistance to blood plasma peptidases inferior to that of PIK1 [12]. These results suggest that all the peptide modifications intended to increase its half-life were successful to a variable extent.…”

“…Among the modifications we implemented in this study, the combined protection of N-and C-termini of L-PIK by the strategies employed in PIK2 and PIK5 proved to be effective and increased the half-life of the abovementioned peptides in human plasma at least 4.5-fold as compared to that of PIK1 [12]. Sure enough, the stability gain of PIK2 and PIK5 was likely to be much higher.…”

“…Previously, we synthesized a set of L-PIK analogues and assessed their proteolytic stability in human plasma in vitro [12]. L-[N α MeArg 1 ]PIK (PIK1) was identified as a leader in these studies because it demonstrated an inhibitory activity similar to that of L-PIK and was about 2.5-fold less susceptible to proteolytic degradation.…”

“…L-[N α MeArg 1 ]PIK (PIK1) was identified as a leader in these studies because it demonstrated an inhibitory activity similar to that of L-PIK and was about 2.5-fold less susceptible to proteolytic degradation. Nevertheless, dipeptidyl carboxypeptidases still degraded PIK1 in plasma [12]. Based on L-PIK and PIK1 degradation patterns, we designed a strategy for development of proteolytically stable L-PIK analogues that surpass PIK1 (summarized in Table 1).…”