Differential Effects of Angelicin Analogues on NF-B Activity and IL-8 Gene Expression in Cystic Fibrosis IB3-1 Cells.

The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor-B (NF-B) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with , the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF-B agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF-B/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF-.

Differential inhibition of signaling pathways by two new imidazo-pyrazoles molecules in fMLF-OMe- and IL8-stimulated human neutrophil.

N-formyl-methionyl-leucyl-phenylalanine (fMLF), its methyl ester fMLF-OMe and interleukin 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, but the mechanisms through which the signal transduction pathways activate this function are not yet completely understood. Compounds 3l and 3r, a new class of arylcarbamoyl-imidazo-pyrazoles derivatives, were described as the first example of compounds able to inhibit human neutrophil chemotaxis induced by both fMLF-OMe and IL8. Here, we report their effects on superoxide production and lysozyme release.

Synthesis and biological evaluation of new active For-Met-Leu-Phe-OMe analogues containing para-substituted Phe residues.

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils.

Effects of synthetic peptides on the inflammatory response and their therapeutic potential.

Recently, interest in small peptide molecules as potential drug candidates has revived. In this review, two series of synthetic peptides and their selective effects on the inflammatory response have been described, focusing on the intracellular pathways involved and on their therapeutic potential. A series of F(D)LF(D)LF analogs has been synthesized, including either N- t-Boc or different N-ureido substituents.

N-Aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides 7-substituted strongly inhibiting both fMLP-OMe- and IL-8-induced human neutrophil chemotaxis.

Anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases. The chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLP), and interleukine 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, probably by two independent mechanisms. We reported here synthesis and biological evaluation of new N-aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides 7-substituted which were designed as possible multi-target antiinflammatory agents.

Anti-inflammatory and analgesic effects displayed by peptides derived from PKI55 protein, an endogenous protein kinase C inhibitor.

We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1-16 (peptide 5), 1-8 (peptide 8) and 1-5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse.

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.

1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carboxylic acid ethyl esters as potent human neutrophil chemotaxis inhibitors.

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis.

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.

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions.

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, betaAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa2 series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa3 series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the betaAla3 15 model reaching the maximum value.

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH.

Elucidation of the involvement of protein kinase C subtypes in several diseases is an important challenge for the future development of new drug targets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lymphocytes and to inhibit the activity of alpha, beta(1) and beta(2) protein kinase C isoforms.

Chemotactic tripeptides incorporating at position 2 alpha-aminoacid residues with unsaturated side chains.

New N-For-Met-Leu-Phe-OMe (fMLF-OMe) analogues incorporating three different gamma-delta-didehydro-alpha-aminoacid residues (namely: Alg = (S)-Allylglycine; Dag = Diallylglycine; Cpg = 1-Aminocyclopent-3-ene-1-carboxylic acid) replacing the native (S)-Leucine have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide fMLF-OMe. Chemotaxis, lysozyme release and superoxide anion production have been measured. (1)H NMR titration experiments and NOESY spectrum of the Cpg containing model 10 have been discussed in order to ascertain the preferred solution conformations.

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr.

New chemotactic dimeric peptides show high affinity and potency at the human formylpeptide receptor.

A number of analogues of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe) have been studied in order to evaluate their ability to interact with formylpeptide receptors and to induce specific biological responses in human neutrophils. In vitro assays were carried out and receptor binding, chemotaxis, superoxide anion release and secretagogue activity were evaluated. The fMLP-OMe analogues synthesized, with the general formula for-Met-Leu-Phe-Xaa-Lys(OMe)-Phe-Leu-Met-for (Xaa=Gly, beta-Ala, gamma-aminobutyric acid, 5-aminovaleric acid, and 6-aminocaproic acid), were constituted by two fMLP units linked by a Lys residue, with an amino acid spacer between them.

2-Phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives: new potent inhibitors of fMLP-induced neutrophil chemotaxis.

It is well known that both acute and chronic autoimmune inflammatory disorders arise following a breakdown in control of neutrophil activation and recruitment. In the search for new anti-inflammatory agents, we synthesized some new 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives and tested them in vitro in order to evaluate their ability to interfere with human neutrophil functions. All tested compounds showed strong inhibition of fMLP-OMe-induced chemotaxis, although they appeared unable to block degranulation and the fMLP-OMe-induced respiratory burst, and were inactive in binding experiments.

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation.

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M.

Synthesis, conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe.

For-Met-betaAlapsi[CSNH]-Phe-OMe (3), For-Met-betaAlapsi[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO(2-Phe-OMe 8a), For-Met-NH-mCH4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. (1)H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated beta-residue.

Structure-activity relationship of for-L-Met L-Leu-L-Phe-OMe analogues in human neutrophils.

Neutrophils constitute the first line of defence against bacterial invasion. They migrate to infected tissues along a concentration gradient of chemoattractant molecules, the most important of which is for-Met-Leu-Phe-OH (fMLP). Different responses arise from formylpeptides binding to different isoforms of the specific receptor.

Hybrid alpha/beta-peptides: for-Met-Leu-Phe-OMe analogues containing geminally disubstituted beta2,2- and beta 3,3-amino acids at the central position.

The two fMLF-OMe analogues For-Met-beta(3)hAc(6)c-Phe-OMe (6) and For-Met-beta(2)hAc(6)c-Phe-OMe (12) and their corresponding N-Boc derivatives 5 and 11 have been synthesized and their biological activity towards human neutrophils evaluated. The N-formyl peptides 6 and 12 exhibit good activity as chemoattractans and 12 is highly active in superoxide anion production. The preferred solution conformation of the two N-formyl derivatives has been discussed.

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.

Identification of CD8+ T cell epitopes within lytic antigens of human herpes virus 8.

The human herpesvirus 8 (HHV-8) is a gamma herpesvirus with oncogenic potential which establishes a chronic infection that is normally controlled by the immune system of healthy individuals. In particular, CTL responses seem to play a key role in control of the infection. In this study, we characterized epitope-specific CTL responses in healthy HHV-8-seropositive individuals against four HHV-8 lytic Ags: open reading frames (ORF) 26, 70, K3, and K5.

Alpha-peptide/beta-sulfonamidopeptide hybrids: analogs of the chemotactic agent for-Met-Leu-Phe-OMe.

In order to gain information on the activity shown by alpha-peptide/beta-sulfonamidopeptide hybrid analogs of the potent chemotactic agent fMLF-OMe, a structure-activity study is reported on N-Boc- and N-formyl tripeptide models containing an aminoalkanesulfonic acid as central residue. Directed migration (chemotaxis), superoxide anion production, and lysozyme release have been measured. The biochemical functions and the conformational properties of the new compounds are discussed and related to previously studied models containing beta-residues.

Chemotactic peptides: fMLF-OMe analogues incorporating proline-methionine chimeras as N-terminal residue.

The new fMLF analogues 1-4, incorporating chimeric S-proline-methionine residues (namely the homochiral cis-4(S)-methylthio-(S)-proline (10) and the heterochiral trans-4(R)-methylthio-(S)-proline) (17) in place of the native S-methionine, have been prepared; their solution conformation and activity as agonists or antagonists of formylpeptide receptors have been studied. In addition to peptides 1-4, which maintain the Met gamma-thiomethyl-ether function, the analogues Boc-PLF-OMe (18) and For-PLF-OMe (19) devoid, as compared with 1-4, of position 1 side chain, have been synthesized and their activity examined.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour.

Objectives: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour.

Methods: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement.

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation.

Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O2-) production from neutrophils.