Synthesis and biological activity of cyclolinopeptide A analogues modified with γ4-bis(homo-phenylalanine)

Abstract

Cyclolinopeptide A (CLA), an immunosuppressive nonapeptide derived from linen seeds, was modified with S or R-γ⁴-bis(homo-phenylalanine) in positions 3 or 4, or both 3 and 4. These modifications changed the flexibility of new analogues and distribution of intramolecular hydrogen bonds. Analogues 11 c(Pro¹-Pro²-Phe³-S-γ⁴-hhPhe⁴-Leu⁵-Ile⁶-Ile⁷-Leu⁸-Val⁹), 13 c(Pro¹-Pro²-S-γ⁴-hhPhe³-R-γ⁴-hhPhe⁴-Leu⁵-Ile⁶-Ile⁷-Leu⁸-Val⁹) and 15 c(Pro¹-Pro²-R-γ⁴-hhPhe³-Phe⁴-Leu⁵-Ile⁶-Ile⁷-Leu⁸-Val⁹) existed as a mixture of stable cis/trans isomers of Pro-Pro peptide bond. The comparison of the relative spatial orientations in crystal state of the two carbonyl groups, neighboring γ-amino acids, revealed conformational similarities to α-peptides. The addition of two -CH₂- groups in γ-amino acids led to a more rigid conformation, although a more flexible one was expected. A significant difference in the relative orientation of the carbonyl groups was found for cyclic γ-peptides with a dominance of an antiparallel arrangement. As carbonyl groups may be engaged in the interactions with plausible receptors through hydrogen bonds, a similar biological activity of the modified peptides was expected. Our biological studies showed that certain cyclic, but not the corresponding linear peptides, lowered the viability of peripheral blood mononuclear cells (PBMC) at 100μg/mL concentration. The proliferation of PBMC induced by phytohemagglutinin A (PHA) was strongly inhibited by cyclic peptides only, in a dose-dependant manner. On the other hand, lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α) production in whole blood cell cultures was inhibited by both linear and cyclic peptides. Peptide 15 c(Pro¹-Pro²-R-γ⁴-hhPhe³-Phe⁴-Leu⁵-Ile⁶-Ile⁷-Leu⁸-Val⁹) blocked the expression of caspase-3, inhibited the expression of caspases-8 and -9 in 24h culture of Jurkat cells, and caused DNA fragmentation in these cells, as an indicator of apoptosis. Thus, we revealed a new mechanism of immunosuppressive action of a nonapeptide.

Keywords: Apoptosis; Caspase; Cyclic peptides; Cyclolinopeptide A; Immunosuppression; PBMC; γ-Amino acids.