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Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.

Cyclolinopeptide A (CLA) is an immunosuppressive peptide of the sequence c-(-Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), isolated from linseed. Since another cyclic, hydrophobic, immunosuppressive peptide, cyclosporin A, has potent antimalarial activity, CLA and a series of its analogues were synthesized on solid phase and tested for inhibition of the human malarial parasite Plasmodium falciparum in culture. The results were compared with the influence of these agents on humoral and cellular immune responses. There was no clear correlation between the structure of the peptides, their immunosuppressive activity, and their antimalarial activity. However, the antimalarial activity of the peptides was apparently connected with the strong hydrophobic nature of CLA. Substitution of a less hydrophobic residue into the peptide chain led to a decrease in or even loss of detectable activity, although such peptides retained the immunosuppressive properties. A possible explanation is that the antimalarial effect of CLA and analogues may result from their influence on cell membranes rather than on some specific receptor such as cyclophilin. In agreement with this idea, binding of CLA to purified P. falciparum cyclophilin was not detected except at very high concentrations. Substitution of D-aromatic residues into the CLA molecule led to a decrease in immunosuppressive activity but had little effect on antimalarial activity, which for these peptides was of the same order as for CLA. We have therefore demonstrated that the cyclolinopeptides are a class of compound not previously shown to have antimalarial activity, and that in a series of analogues there was no correlation between antimalarial and immunosuppressive effects.

The results of the investigation of immunosuppressive activity of cyclolinopeptide A (CLA--cyclic hydrophobic nonapeptide present in the linseeds) and its analogs are discussed. The results obtained for other natural cyclic peptides showing structural similarities with CLA (antamanide, cycloamanides, hymenistatin, hymenamides) are also reviewed. It results from these investigations that the molecular mechanism of the CLA action is the same as that of cyclosporin A and FK-506 compound, i.e. it consists in formation of the complex with cyclophilin and inhibition--in this form--of the phosphatase activity of calcineurin. The results also suggest that the immunosuppressive activity of these compounds resides in their--Pro-Xxx-Phe- fragment, where Xxx is a hydrophobic (e.g. Leu, Val) or aromatic amino acid residue.

The immunosuppressive cyclic nonapeptide cyclolinopeptide A inhibits calcium-dependent, but not calcium-independent, activation of T lymphocytes comparably to the actions of cyclosporin A and FK506. The concentration required for complete inhibition, however, is 10 times higher than that of cyclosporin A. In addition, we demonstrate that calcineurin, a phosphatase which plays an important role in T lymphocyte signalling, is inhibited in vitro by cyclolinopeptide A by a mechanism dependent on the peptidyl-prolyl cis-trans isomerase (PPIase) cyclophilin A but not FKBP12. Direct binding of cyclolinopeptide A to cyclophilin A was confirmed using tryptophan fluorescence studies and PPIase assays. These results represent a third example of the production of a natural product that neutralises calcineurin by a mechanism dependent on the primary binding to a PPIase.

Linear and cyclic analogues of cyclolinopeptide A (CLA) in which one or both phenylalanine residues in fragment Pro6-Pro7-Phe8-Phe9 were substituted by their sulfonated derivatives have been synthesized by SPPS method and cyclization with the BOP reagent. The peptides were examined for their immunosuppressive activity in the humoral and cellular immune response by PFC and DTH tests. All of the analogues retain some immunosuppressive activity of native CLA. Their CD spectra confirm that the optical activity of aromatic residues in CLA depends on their position in the peptide chain. Only the residue in position 8 seems to be optically active. CD spectrum of the cyclic analogue modified in position 9 is very similar to that of native CLA which correlates with its high biological activity. The chiroptical properties of the p-sulfonated Phe-residue are established.

Hymenistatin I (HS-I), a cyclic octapeptide [c-(-Pro-Pro-Tyr-Val-Pro-Leu-Ile-Ile-)], was synthesized by the solid-phase peptide synthesis method and examined for its immunosuppressive activity in the humoral and cellular immune responses. The peptide activity was tested on cell lines producing various cytokines. The results are compared with the activity of the well-known immunosuppressive agent cyclosporin A (CsA). It was found that hymenistatin I exerts immunosuppressive effect (both in the humoral and cellular immune responses) comparable with that of CsA. Comparison of the influence of HS-I and CsA on cytokines production suggests that the mechanisms of the interaction with the immunological system are substantially different for the two compounds tested.

We measured 1H-NMR, fluorescence and CD spectra of cyclolinopeptide A (CLA), its tyrosine analogues with each or both phenylalanines substituted by tyrosine (c-[LeuIleIleLeuValProProTyrPhe], c-[LeuIleIleLeuValProProPheTyr] and c-[LeuIleIleLeuValProProTyrTyr]), and their linear counterparts with the starting sequence leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe (LA). It follows from CD spectra that the conformations of all cyclic peptides are similar to that of CLA; the conformations of linear peptides are more diversified, with the conformation of [Tyr9]LA being most similar to CLA. NMR studies suggest that aromatic rings in cyclic peptides are situated perpendicular to each other, manifesting edge-to-face pairing. Accordingly, the residue in position 9 is shielded ('edge'), and a residue in position 8 is the shielding one ('face'). This effect is not present in the case of linear peptides. Fluorescence quantum yields were much lower for cyclic peptides than for linear ones, indicating the interaction of closely located aromatic chromophores. Those quantum yields depend on the relative position of Tyr in the peptide chain. Another factor influenced by the position in the peptide chain is the optical activity of aromatic side chains (optically active in position 8, inactive in position 9). This phenomenon could be explained by the differences in the side-chain conformation of both aromatic residues.

We demonstrated that a linear peptide Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe, the analog of a cyclic nonapeptide c-(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe) possesses a distinct immunosuppressive activity. We synthesized a series of eight other analogs of the linear peptide. The series consists of the nonapeptides protected on N- and/or C-terminus by acetyl-, succinyl-, and amido- functions and of Gly-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe elongated on N- and/or C-terminus by additional glycine residues. It was found that the protection of the terminal functional groups of the peptide slightly increases its immunosuppressor potency. At the same time the elongation of the peptide chain on both termini by additional glycine residues evokes a distinct increase of the immunosuppressive activity.