fMLP-OMe analogues trigger specific signalling pathways in the physiological functions of human neutrophils

Human neutrophils are phagocytic cells involved in host defence mechanisms against bacterial infections. It has been demonstrated that small formyl-peptide derivatives, obtained as bacterial metabolites or derived from disrupted mitochondria, can be potent chemoattractants for phagocytes. for-Met-Leu-Phe (fMLP), together with its synthetic methyl ester derivative for-Met-Leu-Phe-OMe (fMLP-OMe), is used as a model chemoattractant due to its highly effective ability to activate all physiological functions of neutrophils through binding with specific G-protein coupled receptors (FPR) located on the neutrophil membrane. The interaction of fMLP with its receptor expressed on neutrophils triggers multiple second messengers through the activation of phospholipase C (PLC), PLD and PLA2 and rapidly stimulates phosphatidylinositol-3-kinase (PI3-K), as well as activating tyrosine phosphorylation. An increase in intracellular levels of cyclic AMP (cAMP) and the involvement of kinases, such as protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) [Jun N-terminal kinases (JNK), p38 and extracellular response kinases 1 and 2 (ERK1/2)], has also been demonstrated. The activation of these transduction pathways is known to be responsible for various biochemical responses which contribute to the physiological defence against bacterial infections and cell disruption. In fact, it has long been known that the transduction pathway underlying the chemotactic response is different from those responsible for cytotoxic functions, and several previous experiments carried out utilizing pharmacological manipulation of the signal transduction pathway have highlighted the fact that distinct mechanisms are involved in each of these neutrophil responses. This can be rationalized on the basis of the existence of at least two different functional receptor subtypes or isoforms; low doses of a full agonist (or a "pure" chemoattractant) are required to interact with a high-affinity receptor subtype (FPR) that activates the transduction pathway responsible for the chemotactic response, while the increase of the full agonist concentration - typical of infections sites - allows binding with the its low-affinity subtype (FPRLike-1), able to activate the transduction pathways responsible for superoxide anion production and lysozyme release. The use of selective analogues (ligands able to discriminate between different biological responses) allowed us to confirm the idea that fine tuning of neutrophil activation occurs through differences in activation of a spectrum of signalling pathways. For each stimulus capable of a unique set of cellular responses, a distinctive imprint of signal protein activation may exist. Through more complete understanding of intracellular signalling, new drugs could be developed for the selective inflammatory blockade

Oligomeric formylpeptide activity on human neutrophils

A series of oligomeric formylpeptides were synthesized by cross-linking the prototype fMLP using a Lys residue. They were then investigated for their ability to stimulate chemotaxis, superoxide anion production, and lytic enzyme release in human neutrophils. Although active in stimulating the different receptor isoforms, leading to the different biological responses, these analogues showed a lesser potency and affinity than the standard peptide. On the basis of the results reported here, we can hypothesise that: (i) the increased bulk of these molecules seems to hinder their correct allocation into the receptor pocket, thereby hindering favourable receptor interaction; and that: (ii) fMLP space positions do not seem to allow the ligand to increase biological responses

Signal transduction pathways triggered by selective formylpeptide analogues in human neutrophils

Human neutrophils are highly specialised for their primary function, i.e. phagocytosis and destruction of microorganisms. Leukocyte recruitment to sites of inflammation and infection is dependent upon the presence of a gradient of locally produced chemotactic factors. The bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was one of the first of these to be identified and is a highly potent leukocyte chemoattractant. It interacts with its receptor on the neutrophil membrane, activating these cells through a G-protein-coupled pathway. Two functional fMLP receptors have thus far been cloned and characterized, namely FPR (formyl peptide receptor) and FPRL1 (FPR like-1), with high and low affinities for fMLP, respectively. FMLP is known to activate phospholipase C (PLC), PLD, PLA2 and phosphatidylinositol-3-kinase (PI3K), and it also activates tyrosine phosphorylation

Properties of a novel chemotactic esapeptide, an analogue of the prototypical N-formylmethionyl peptide

The new disulphur-bridged peptide, for-Met-Leu-Cys(OMe)-Cys(OMe)-Leu-Met-for, has been synthesized and its biological properties resulting from its binding to the formyl-peptide receptor of human neutrophils characterized. Three activities resulting from this interaction were measured: directed cell migration (i.e., chemotaxis); superoxide anion production; and lysozyme enzyme release. The properties were compared with those observed for the prototypical peptide, for-Met-Leu-Phe-OMe. Chemotaxis is strong1y triggered while both superoxide anion production and lysosomal enzyme release are elicited only at high concentrations and never reach the response peak observed for the prototype peptide at physiologically relevant concentrations. The derivative appears to bind with a good affinity to the formyl-peptide receptors. These results provide new information regarding the structure~activity relationship of the formyl-peptide receptor

Novel chemotactic For-Met-Leu-Phe-OMe (fMLF-OMe) analogues based on Met residue replacement by 4-amino-proline scaffold: Synthesis and bioactivity

cis-(2S,4S) 4-Amino-proline (cAmp) and trans-(2S,4R) 4-amino-proline (tAmp) residues, bearing N-For or N-Boc substituents at the two amino groups, have been incorporated into the potent chemotactic agent fMLF-OMe in place of the N-terminal native (S)-methionine to give the analogues 17a–19a and 17b–19b. The new ligands have been examined for their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils as agonists and antagonists. Compounds 19a and 19b, bearing two N-For groups at the proline scaffold, are active and selective chemoattractants. The ligand 18b, containing N-For at the 4-amino group of the N-Boc-tAmp residue, exhibits significant chemotactic antagonism. The influence of the different substitution at the N-terminal position of the new analogues is discussed

Studies on human neutrophil biological functions by means of formylpeptide receptor agonists and antagonists

Phagocytes are activated by several extracellular signals, including formylpeptides 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-Lphenylalanine (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