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. A fully extended (C(5)) conformation at position 2 and a folded conformation with two consecutive gamma-turns (C(7) structure) have been proposed for the Dag and Cpg containing tripeptides, respectively.

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.

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.

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. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2- from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22)>Ro 64-6198 (pEC50 12.96)>N/OFQ(1-13)NH2 (pEC50 12.67)>N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 microM while antagonized by UFP-101 10 microM (pA2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.

Studies on human neutrophil biological functions by means of formyl-peptide receptor agonists and antagonists.

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.

Biological variation responses in fMLP-OMe analogs, introducing bulky protecting groups on the side-chain of hydrophilic residues at position 2.

for-Met-Ser(Bzl)-Phe-OMe, for-Met-Cys(Bzl)-Phe-OMe, for-Met-Tyr(Bzl)-Phe-OMe and for-Met-Lys(Z)-Phe-OMe were synthesized to investigate the importance of a bulky protecting group on the side-chain of a hydrophilic residue at position 2 on the biological activities of human neutrophils. Our results indicate that these compounds do not trigger a good chemotactic response, which, in any case, is not improved with respect to that induced by the analogs with the unprotected residues. Instead, both superoxide anion production and, particularly, lysozyme release are more efficient.

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.

Biological activity in human neutrophils of di-tripeptides, analogues of the chemotactic fMLP.

for-Met-Ser(for-Met-Leu-Phe)-Phe-OMe 1 and for-Met-Lys(for-Met-Leu-Phe)-Phe-OMe 2 were synthesized in order to investigate biological activities on human neutrophils of crosslinked di-tripeptides. Our results seem to highlight that the tested di-tripeptides (i) do not step up chemotaxis, (ii) can elicit superoxide anion production which is dependent on the nature of the residue at position 2, chosen in the tripeptide that is crosslinked to the fMLP-OMe.

A hydrophilic residue at position 2 can improve specific biological responses in fMLP-OMe analogs.

The peptides for-Met-Ser-Phe-OMe 1, for-Met-Cys-Phe-OMe 2, for-Met-Lys-Phe-OMe 3, and for-Met-Tyr-Phe-OMe 4 were synthesized in order to investigate the importance of a hydrophilic side-chain on the residue at position 2 on biological activities of human neutrophils. Our results seem to highlight that this type of substitution does not facilitate good chemotaxis, although it elicits both superoxide anion production and particularly lysozyme release, in some cases even more potent than the parent fMLP-OMe, if the hydrophilicity is associated with steric hindrance.

Inhibition of neutrophil responses by cyclosporin A. An insight into molecular mechanisms.

OBJECTIVE: Cyclosporin A (CsA) is an effective agent in rheumatoid arthritis (RA), slowing joint damage progression. Its therapeutic effect on T lymphocytes has been studied extensively, but there is little information available about neutrophils, the cells responsible for a substantial proportion of inflammation. A study was performed to investigate the in vitro effects of CsA on neutrophil functions triggered by several agonists and determine whether the drug could counteract the binding of formyl-methionyl-leucyl-phenylalanine (fMLP) to its receptor and/or modulate changes in the intracellular Ca(2+) concentration ([Ca(2+)]i). METHODS: CsA was added to neutrophils 5-50 min before the incubation steps for neutrophil function assays (chemotaxis, superoxide anion production, lysozyme release), calcium measurements and receptor binding experiments. RESULTS: CsA appeared to be particularly effective in lowering chemotaxis, superoxide anion production and lysozyme release induced by different agonists. However, it did not significantly affect either basal or agonist-stimulated neutrophil [Ca(2+)]i and the interaction between fMLP and its receptor. CONCLUSIONS: Because of its in vitro inhibition of neutrophil functions, CsA appears to have considerable potential as an anti-inflammatory drug. Moreover, as it is also a potent immunosuppressive agent, it may reduce the progression of joint damage in RA. More work remains to be done to clarify the molecular mechanism of CsA action on neutrophils.

Pseudopeptides containing the 2-hydrazonoacyl fragment: analogs of the chemotactic agent HCO-Met-Leu-Phe-OMe.

In order to further examine the properties of pseudopeptides containing the 2-hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N-formyl-tripeptide HCO-Met-Leu-Phe-OMe were designed and synthesized. The first group contains the new fragment as the N-terminal residue and is represented by the N-aryl derivatives p-Cl-C6H4-NH-N=C(R)-CO-Leu-Phe-OMe (2 and 3) and by the corresponding N-aroyl analogs p-CH3-C6H4-CO-NH-N=C(R)-CO-Leu-Phe-OMe (4). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO-Xaa-NH-N=C(R)-CO-Phe-OMe (7a and 7b) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.

Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues.

The N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-OMe (1) analogues for-Thp-Leu-Ain-OMe (2), for-Thp-Leu-Phe-OMe (3), for-Met-Leu-Ain-OMe (4), for-Met-Delta(z)Leu-Phe-OMe (5), for-Met-Lys-Phe-For-Met-Lys-Phe (6), for-Met-Leu-Pheol-COMe (7), and for-Nle-Leu-Phe-OMe (8) have been studied. Some of these have been found selective towards the activation of different biological responses of human neutrophils. In particular, peptides 2 and 3, which evoke only chemotaxis, are ineffective in enhancing inositol phosphate, as well as cyclic AMP (cAMP) levels. On the contrary, analogues 5 and 7, which induce superoxide anion production and degranulation, but not chemotaxis, significantly increase the levels of the two intracellular messengers, as is the case of the full agonists 1 and 6. The Ca(2+) ionophore A23187 also activates phospholipase C (PLC) and increases the nucleotide levels; when tested in combination with peptide 1 or 5, a supra-additive enhancement of cAMP concentration is obtained. The PLC blocker, U-73122, inhibits the formylpeptide-induced inositol phosphate formation, as well as cAMP increase. Moreover, this drug drastically reduces superoxide anion release triggered by 1 or 5, whereas it inhibits to a much lesser extent neutrophil chemotaxis induced by 1 or 2. Our results suggest that: (i) PLC stimulation is involved in cAMP enhancement by formylpeptides; (ii) the activation of PLC by formylpeptides, in conditions of increased Ca(2+) influx, induces a supra-additive enhancement of the nucleotide; (iii) the inability of pure chemoattractants to significantly alter the PLC activity or cAMP level, differently from full agonists or peptides specific in inducing superoxide anion release, appears as a general property. Thus, the activation of neutrophil PLC seems essential for superoxide anion release, but less involved in the chemotactic response.

Met-Ile-Phe-Leu derivatives: full and partial agonists of human neutrophil formylpeptide receptors.

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.

Synthesis and activity on human neutrophil functions of fMLF-OMe analogs containing alkyl spacers at the central position.

We report here the synthesis and activity of new analogs of the N-formyl and N-tert-butyloxycarbonyl (Boc) derivatives of the tripeptide Met-Leu-Phe-OMe containing an achiral omega-amino acid residue replacing the hydrophobic central leucine. The tripeptides HCO-Met-NH-(CH2)n-CO-Phe-OMe and Boc-Met-NH-(CH2)n-CO-Phe-OMe (n = 3-5) containing the central homomorphic residue of 5-aminopentanoic acid (delta-aminovaleric acid; delta-Ava; n = 4) and the two non-homomorphic residues of 4-aminobutanoic acid (gamma-aminobutyric acid; gamma-Abu; n = 3) and 6-aminohexanoic acid (epsilon-aminocaproic acid; epsilon-Aca; n = 5) have been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the gamma-Abu and the delta-Ava residues, show good and selective antagonist activity on superoxide anion production.

Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.

Biscarbamate analogues of the chemotactic tripeptide fMLF-OMe.

Based on the sequence of the prototypical chemotactic tripeptide HCO-Met-Leu-Phe-OH (fMLF) and by taking into account the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCO-Met-Leu-gPhe-COOMe (2) and Boc-Met-Leu-gPhe-COOMe (4) were synthesized. These two new ligands are characterized by the presence of a gem-diamino residue (gPhe) replacing the C-terminal Phe and a carbamate functionality positioned at both the ends of the molecule. The activity of the two new compounds has been determined on human neutrophils and compared to that shown by the corresponding N-terminal monocarbamates MeOCO-Met-Leu-Phe-OMe (1) and Boc-Met-Leu-Phe-OMe (3).

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues.

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.

Bioactive fMLF-OMe analogs containing a N-terminal oximic or formylhydrazonic moiety.

In order to obtain chemotactic peptides with selective bioactivity, a new type of structural modification was introduced at the N-terminal position of HCO-Nle-Leu-Phe-OMe. Two groups of analogs have been synthesized both containing a N-terminal residue of the X=C(R)-CO-type replacing the native HCO-NH-CH(R)-CO-. In particular, the A group of pseudopeptides (2a-d) possesses a N-terminal oximic fragment (X=HO-N) and the B group (3a-d) a formylhydrazone fragment (X=HCO-NH-N). These new ligands have been examined for their capacity to induce chemotaxis and other cellular responses such as superoxide anion production and lysozyme release; although significantly active as chemoattractants they have been found to be practically devoid of secretagog activity, thus exhibiting selective behavior. The adopted chemical modification seems extensible in designing a new class of pseudopeptides (hydrazonopeptides) structurally related to both hydrazinopeptides and peptides containing alpha,beta-unsaturated residues.

Peptide T-araC conjugates: solid-phase synthesis and biological activity of N4-(acylpeptidyl)-araC.

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.