S-(γ)-Phe

Cyclolinopeptide A, naturally occurring immunomodulatory nonapeptide, was modified with S or R-γ(3)-bis(homophenylalanine) in positions 3 or 4, or both 3 and 4. The replacement of one or both Phe residues by γ(3)-hhPhe led to decrease of their conformational flexibility in the analogues in comparison to CLA. All cyclic peptides, except 11, exist as isomers with the cis Pro-Pro peptide bond. Cyclic peptide 11 with single modification S-γ(3)-hhPhe(4) exists as a mixture of two isomers and the major isomer (89%) contains all peptide bonds of the trans geometry. The peptides were subjected to several immunological tests in vitro and in vivo. Linear peptides 1-8, precursors of CLA analogues 9-16, were not toxic against human peripheral blood mononuclear cells (PBMC) but cyclic analogues showed dose-dependent toxicity with exception of peptide 11. Linear peptides did not inhibit mitogen-induced PBMC proliferation whereas cyclic ones inhibited the proliferation in a dose-dependent manner. The actions of linear and cyclic peptides with regard to lipopolysaccharide (LPS) -induced tumour necrosis factor alpha (TNF α) production in whole human blood cultures were differential but particularly suppressive in the case of linear compound 6. Therefore, for in vivo tests compounds 6 and 11 were selected. The compounds showed comparable, suppressive actions in induction and effector phases of delayed type hypersensitivity as well as in the carrageenan-induced foot pad edema in mouse models. In summary, linear peptide 6 and cyclic peptide 11 are attractive as potential immune suppressor drugs.

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.

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.