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Phagocytes are activated by several extracellular signals, including formyl-peptides derived from bacterial proteins or disrupted cells. The most intensely studied member of the formylpeptide family is the synthetic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), whose specific receptors have been identified on neutrophil plasma membrane and subsequently cloned. The fMLP-receptor interaction activates multiple transduction pathways responsible for various neutrophil functions such as adhesion, chemotaxis, exocytosis of secretory granules and superoxide anion production, which represent the physiological response to bacterial infection and tissue damage. An unresolved question is whether signaling requirements are identical or specific for each physiological function. The development of fMLP receptor agonists and antagonists has led to an improvement of our knowledge about the above issue. Of particular interest is the possibility that receptorial antagonists, able to transiently inhibit neutrophil responses to formylpeptides, could be therapeutic agents in the treatment of inflammation-related diseases. Aim of this review is, i) to summarise the current understanding of the series of events that begins at the level of formylpeptide-receptor interaction and is responsible for the activation of transduction pathways, which finally determine neutrophil response; ii) to define the state of art regarding the synthesis as well as the biological actions of fMLP receptor agonists and antagonists.

The biological action of several X-Phe-D-Leu-Phe-D-Leu-Z (X=3',5'-dimethylphenyl-ureido; Z=Phe, Lys, Glu, Tyr) analogues was analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-D-Leu-Phe-D-Leu-Phe analogues obtained as C-terminal olo or amido derivatives and T-Phe-D-Leu-Phe-D-Leu-Phe analogues (T=thiazolyl-ureido) were also analysed. The activities of pentapeptide derivatives were compared with those of X-Phe-D-Leu-Phe-D-Leu-Phe chosen as reference antagonist. Our results demonstrate that X-Phe-D-Leu-Phe-D-Leu-Phe-olo, X-Phe-D-Leu-Phe-D-Leu-Glu and X-Phe-D-Leu-Phe-D-Leu-Tyr are more active antagonists than X-Phe-D-Leu-Phe-D-Leu-Phe. The presence of Lys (X-Phe-D-Leu-Phe-D-Leu-Lys) seems, instead, to inhibit the formyl-peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor antagonist properties of T-Phe-D-Leu-Phe-D-Leu-Phe-OH. The introduction of the C-terminal methyl ester (T-Phe-D-Leu-Phe-D-Leu-Phe-OMe) or amido group (X-Phe-D-Leu-Phe-D-Leu-Phe-NH2) appears detrimental for the affinity and formyl-peptide receptor antagonist properties of the Phe-D-Leu-Phe-D-Leu-Phe derivatives. The examined peptides inhibit superoxide anion production and lysozyme release more efficaciously than neutrophil chemotaxis.

The conformationally constrained f-L-Met-Ac(n)c-L-Phe-OMe (n = 4,9-12) tripeptides, analogues of the chemoattractant f-L-Met-L-Leu-L-Phe-OH, were synthesized in solution by classical methods and fully characterized. These compounds and the published f-L-Met-Xxx-L-Phe-OMe (Xxx = Aib and Ac(n)c where n = 3, 5-8) analogues were compared to determine the combined effect of backbone preferred conformation and side-chain bulkiness at position 2 on the relation of 3D-structure to biological activity. A conformational study of all the analogues was performed in solution by FT-IR absorption and 1H-NMR techniques. In parallel, each peptide was tested for its ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The biological and conformational data are discussed in relation to the proposed model of the chemotactic receptor on neutrophils, in particular of the hydrophobic pocket accommodating residue 2 of the tripeptide.

The biological action of a series of Met-Ile-Phe-Leu analogues was analyzed on human neutrophils, to evaluate their ability to interact with formylpeptide receptors and to induce the related neutrophil responses. Three in vitro assays were carried out: receptor binding, chemotaxis and superoxide anion release. Our results demonstrate that formyl-Met-Ile-Phe-Leu derivatives act as more potent full agonists than formyl-Met-Leu-Phe, the tripeptide normally used as a model chemoattractant for the study of cell functions. On the other hand, the presence of N-ureidoisopropyl substituent in tetrapeptides imparts weak partial agonist properties. It has furthermore been demonstrated that the C-terminal methyl esterification or amination weakly influences the properties of tetrapeptide homologues. Finally, t-Boc-Met-Ile-Phe-Leu derivatives do not appear able to interact with formylpeptide receptors.

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.

The conformation of several Phe-D-Leu-Phe-D-Leu-Phe analogues was analyzed using infrared absorption and circular dichroism. Their effect on human neutrophils was verified by receptor binding and chemotaxis assays. The results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipatic environment, and that they are able to antagonize the neutrophil functions evoked by CHO-Met-Leu-Phe.

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.

In order to obtain an insight into the mode of binding at the for-Met-Leu-Phe-OH (fMLP) receptor, three fMLP-OMe analogs (1-3) were synthesized in which the Met residue was substituted by Gln 1, Asn 2, and Ser 3. We evaluated the influence of the charge variation and/or the shift of its position on neutrophil biological responses.