Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides.

Six retro-inverso tri- and tetrapeptide analogues of RGD were prepared and their anti-aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl-aspartic residue into a peptide chain is described for the first time. A 2-3-fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure-activity relationships are discussed.

Chemical and enzymatic treatment of endothelin.

Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containing 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7-NH2]-7,8-seco-ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated alpha-amino group of the Asp8 residue. The Lys9-Glu10 bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9-Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13,14-seco-ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives.

D-amino acid scan of endothelin: importance of amino acids adjacent to cysteinyl residues in isomeric selectivity.

A systematic approach to map the functionally important determinants of endothelin-1 (ET-1) by a D-amino acid scan is described. Correct orientation of the amino acid side chains was generally of paramount importance both for binding at the ETA receptor and for contracting activity. This was particularly valid for positions 2, 8, 14, 16-21 (the four Cys residues were kept unaltered). Nevertheless, increment of binding affinity was observed by inversion of configuration at positions 6, 7, 9 and 10. In addition, [D-Lys9]ET was an agonist about four times more potent than the natural compound. Usually both 1,4- and 1,3-isomers (corresponding, respectively, to the correct and misfolded disulfide bridges of ET) were obtained, and usually the isomer formed in larger amount had the higher HPLC retention time and the higher biological activity. However, four out of seventeen single-point D-amino acid analogues could be isolated only in one isomeric form. In three cases (D-Ser2, D-Ser4, D-Val12), the inverted amino acid was adjacent to a Cys residue, and in one case (D-Lys9) it was one amino acid apart, thus suggesting a possible effect of the bridged cysteinyl residues in isomeric selectivity.

Role of positions 9 and 10 in the endothelin molecule for biological activity and discrimination of receptor subtypes.

The importance of residues 9 and 10 in endothelin-1 was assessed by studying the responses of the guinea-pig ileum to [Ala9]endothelin-1 and [Ala10]endothelin-1. Both analogues induced relaxation followed by contraction. [Ala9]Endothelin-1 showed similar ED50 values and maximum response to those of endothelin-1, whereas [Ala10]endothelin-1 showed a larger ED50 value and was a partial agonist. Endothelin-1 and [Ala10]endothelin-1 induced similar degrees of tachyphylaxis, whereas [Ala9]endothelin-1 induced very little tachyphylaxis, indicating that Lys9 is important for inducing tachyphylaxis. Apamin inhibited the relaxation induced by endothelin-1 and [Ala9]endothelin-1 but not that induced by [Ala10]endothelin-1. BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu), a specific endothelin ETA receptor antagonist, inhibited [Ala9]endothelin-1-, but not [Ala10]endothelin-1-induced contraction. Cross-tachyphylaxis and additivity studies indicated that [Ala9]endothelin-1, like endothelin-1, acts at the endothelin ETA receptor, whereas [Ala10]endothelin-1 behaved as an endothelin ETB receptor agonist, like sarafotoxin S6c. Thus, the residue at position 10 plays a significant role in receptor activation and is a candidate for further exploration of receptor antagonism.

Structure-activity relationships of endothelin-1 analogues.

The importance of residues 9 and 10 for endothelin-1 (ET-1) biologic activity was assessed by studying the responses of the guinea pig ileum to [Ala9]-ET-1 and [Ala10]-ET-1. Both analogues induced relaxation followed by contraction. [Ala9]-ET-1 showed similar ED50 value and maximal response to those of ET-1, whereas [Ala10]-ET-1 had a larger ED50 value and was a partial agonist, as was IRL1620. ET-1 and [Ala10]-ET-1 induced similar degrees of tachyphylaxis, whereas [Ala9]-ET-1 induced very little tachyphylaxis, indicating that Lys9 is important for inducing tachyphylaxis. BQ-123, an ETA antagonist, did not inhibit the relaxation. It did inhibit [Ala9]-ET-1- and ET-1-induced contractions but not [Ala10]-ET-1- and IRL1620-induced contractions. Cross-tachyphylaxis and additivity studies indicated that [Ala9]-ET-1, like ET-1, acts at the ETA receptor, whereas [Ala10]-ET-1 behaved as an ETB receptor agonist, like sarafotoxin S6c. Therefore, the residue at position 10 plays a significant role in receptor activation and is a candidate for further exploration of receptor antagonism. FCE27037 (cyclo [D-Cys11-Cys15]-ET-1[8-21]) inhibited the contractile but not the relaxant component of the response induced by IRL1620. These results indicate that FCE27037 is a new ETB antagonist and a useful tool that can discriminate pharmacologically the functionally distinct ETB receptors present in the guinea pig ileum.

Side-reactions in peptide synthesis. 2. Formation of by-products in the large-scale synthesis of THF-gamma 2 by solution synthesis.

The impurities which formed in the large-scale synthesis of THF-gamma 2, an immunomodulatory peptide of formula H-Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu-OH, were identified by a combination of analytical methods, and their structure confirmed by synthesis. Most impurities originated from side-reactions involving the aspartyl residue (cyclization, beta-aspartyl formation and cleavage). Based on this knowledge, modifications were introduced into the work up and the purification procedure which resulted in a very pure final product (> 99% by RP-HPLC).

Alanine scan of endothelin: importance of aromatic residues.

A systematic approach to map the functional important determinants of endothelin-1 (ET) by an alanine scan is described. Studies on the in vitro receptor binding affinity and on the agonist contracting activity defined that residues Asp8, Tyr13, Phe14, Leu17, and Trp21 were of major biological significance. A striking observation was that four out of these five sites were hydrophobic amino acids. Ala analogues of the aromatic residues at position 13, 14, and 21 displayed sharply reduced receptor binding affinity (< 2% of ET) and can be considered important for receptor contact. Ala analogues of Asp8 and Leu17 lost most (> 90%) of the agonist activity but retained a receptor affinity nearly equivalent to ET and can be considered to be important for signal transduction. Three other positions, Val12, Asp18, and Ile20 (which are adjacent to the biologically important sites of Tyr13, Leu17, and Trp21), resulted as partially tolerant to Ala substitution, retaining 14-50% of the potency of ET. Ala analogues of the Et isomeric disulfide arrangement (Cys1,11 and Cys3,15) were always less active than the corresponding analogues with the native disulfide pairings (Cys1,15 and Cys3,11).

In vitro and in vivo activity of alkylating bombesin receptor antagonists on small cell lung carcinoma.

Bombesin (BN) and bombesin-like peptides are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. Six peptides belonging to a new class of alkylating BN analogues have been selected according to their characteristics evidenced on Swiss 3T3 fibroblasts: high binding affinity to BN receptor, relevant inhibition (> 60%) of the proliferative stimulus induced by BN, long-lasting effect and specificity for BN receptor. The six peptides were able to bind BN receptors on SCLC cells and to inhibit the growth of two SCLC cell lines: NCI-H69 and NCI-N592. Conversely, they did not inhibit the growth of tumor cell lines devoid of BN receptors. Two of them were tested in vivo on N592 cells transplanted into nude mice. The peptide carrying a Cab [p-bis(2 chloroethyl)aminobenzoyl] moiety proved to be completely inactive. The second peptide, with a Melphalan moiety (Mel), showed a moderate activity (33-45% of tumor growth inhibition) without any toxicity. The low solubility of this compound prevented the use of the higher doses in vivo.

Bombesin receptor antagonists. 5 new irreversible alkylating analogues.

New alkylating bombesin analogues were synthesized in order to increase their solubility and stability in aqueous solutions. The best compromise between these parameters and the biological properties (receptor binding and antagonistic activity) was achieved with 4-[bis(2-chloro-ethylamino)]benzoyl derivatives of the BN (7-14) octapeptide carrying a (13-14) reduced peptide bond independently of the presence of a His12 residue, either free or protected.

Bombesin receptor antagonists. 4. Chloromethylketone and chloroethylnitrosourea derivatives.

Four alkylating bombesin (BN) analogues (two C-terminal and one N-terminal chloromethylketone derivatives, and one chloroethylnitrosourea derivative) were synthesized and tested in Swiss 3T3 fibroblasts for receptor binding and mitogenic activity. Although they bound to the BN receptor with no or very weak mitogenic activity, no one analogue inhibited BN-induced thymidine incorporation in the contemporaneous treatment; only one behaved as a weak receptor antagonist when given 24 h before BN stimulation.

Conformational studies on bombesin antagonists: CD and NMR characterization of [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14).

The conformational flexibility of the [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14) nonapeptide has been studied by CD and one- and two-dimensional (1D and 2D) nmr techniques. The CD and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for the parent bombesin (BN) and [D-Phe12, Leu14]BN. A preliminary investigation on spantide is also reported. In particular, the results obtained from CD measurements indicate that there is a shift from random coil structures, in aqueous solutions, toward folded structures in apolar media (2,2,2-trifluoroethanol) and in a membrane-mimetic environment (40 mM SDS) for all three peptides, namely BN, [D-Phe12, Leu14]BN, and [Thr6, Leu13 psi(CH2NH) Met14]BN (6-14). Spantide, which also possesses some inhibitory activity against BN but very little sequence similarity, even in water, shows an ordered conformation. Nuclear magnetic resonance parameters such as backbone NH-alpha CH coupling constant values, amidic temperature coefficients, and the presence of only sequential nuclear Overhauser effects have not provided, so far, any clear evidence for a preferential ordered structure in the peptides studied, and this may be due to rapid exchange among different conformers in the nmr time scale.

Bombesin receptor antagonists. 2. Analogues based on substance P antagonists.

Although bombesin (BN) and substance P share only the C-terminal dipeptide amide, some substance P receptor antagonists are also weak bombesin receptor antagonists. In order to increase the selectivity of the antagonism for the BN receptor, a series of hybrid peptides were synthesized by the solid-phase methodology, and screened on 3T3 fibroblasts for binding and mitogenic activity. The analogues inhibiting BN-induced thymidine incorporation were further tested for peripheral (amylase release and urinary bladder contraction) and central activity (grooming behaviour).

Bombesin receptor antagonists. 3. Irreversible alkylating analogues: melphalan derivatives.

Bombesin (BN)-like peptides (such as GRP, gastrin-releasing peptide) are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. The present paper deals with a new class of bombesin analogues carrying a nitrogen mustard at their N-terminus. Due to the irreversible binding to the BN receptor(s), these peptides eventually block the mitogenic effects of the natural ligand(s), regardless of their intrinsic "agonistic" or "antagonistic" structures. In Swiss 3T3 fibroblasts they inhibit [125I]GRP binding in the nanomolar/micromolar range. According to their "agonistic" or "antagonistic" structural features, they do or do not induce [3H]thymidine incorporation and p 115 phosphorylation. In competition experiments, alkylating "antagonists" selectively inhibit BN-induced thymidine incorporation either when given simultaneously with or 24 hours before the BN challenge. Alkylating "agonists" display antagonistic effects only in the sequential treatment.

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Fluorescence, CD, and NMR studies on spantide, a bombesin and substance P antagonist.

The conformational properties of spantide [(D-Arg, D-Trp, Leu) substance P] have been studied by fluorescence, CD, and nmr techniques. The fluorescence, CD, and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for bombesin. A preliminary investigation on [D-Pro] spantide is also reported.

Caerulein receptors on the cholinergic neurons in guinea-pig ileum.

The release of [3H]ACh and the contractions of guinea-pig ileal longitudinal muscle preparations, with myenteric plexus attached were measured and recorded simultaneously. Caerulein in concentrations of 10(-11) M to 10(-8) M caused dose-dependent increase of the contractions and the [3H]ACh release. This excitatory effect of caerulein was sensitive to TTX (10(-6) M). Proglumide selectively antagonized both the contractions and the [3H]ACh-releasing effect of caerulein. Electrical field stimulation (O.1 Hz)-evoked contractions were sensitive to atropine. Caerulein (10(-9) M) did not influence the electrically-evoked release of [3H]ACh and the electrically-evoked contractions. Nifedipine (10(-6)M) decreased them about 50%. In the presence of nifedipine caerulein produced an inhibitory effect on the electrically-induced contractions. This nifedipine-unmasking inhibitory effect of caerulein was accompanied by a decrease of the [3H]ACh release and was prevented by proglumide.

Bombesin receptor antagonists [corrected]. 1. Analogues with deleted, inverted or substituted amino acid residues.

Bombesin is a 14-residue peptide hormone which serves a variety of biological functions, including cell growth control in Swiss 3T3 cultured fibroblasts. It has been also involved in an autocrine system regulating the growth of small cell lung carcinoma. A series of bombesin analogues were developed by amino acid deletion, inversion or substitution in the carboxy-terminal region, identified by the target cell receptor. Their properties were examined for: i) competitive binding assays; ii) mitogenic induction and inhibition assays; iii) effects on early cellular events (inositol phosphates production and protein tyrosine phosphorylation). Inversion of the Trp residue, or deletion of the C-terminal tripeptide amide, induced complete loss of receptor affinity and biological effects. Deletion of the His residue, or its derivatization as His (Dnp) in conjunction with Met deletion or modification, gave rise to compounds with reduced receptor affinity and weak antagonistic properties. Other modifications in the C-terminal tripeptide affected the potency but not the biological profile of the parent peptide. These results indicate the basis for the eventual design of improved, specific bombesin receptor antagonists and stimulate further studies on their possible application in the therapy of human small cell lung cancer.

Dermorphin interaction with rat brain opioid receptors: involvement of hydrophobic sites in the binding domain.

Dermorphin structural analogues were utilized to determine the nature of opioid receptor subsite specificity, affinity, and selectivity in rat brain membranes. The data suggest that these parameters are influenced by the amino acid composition and sequence and the known solution conformation of dermorphin, in addition to the conformation of the membrane receptor. Two hydrophobic components of dermorphin are required for optimal binding. One component encompasses the stacked phenol groups in Tyr1 and Tyr5; the second involves the phenyl group of Phe3. Evidence to support this proposal includes the following results: (a) removal of aromaticity, as occurs in [des-Tyr5]- and [Gly5]dermorphin, drastically reduced binding to both mu and delta sites; (b) inversion of the Phe3-Gly4 sequence in dermorphin to the Gly3-Phe4 in enkephalin enhanced binding to delta receptor sites, yet the peptide remained mu-selective; (c) substitution of Pro4 for Gly4 disrupted the solution conformation of dermorphin and decreased affinities at both receptor subsites, substantiating the requirement for the Phe3-Gly4-Tyr5 sequence in dermorphin to interact with mu sites; and (d) modification of the serine residue, as occurs in [Ser(Bzl7)] dermorphin and [Ser-NHNHZ7]dermorphin, enhanced interaction with delta opioid receptors; the apparent delta affinity increased over 50-fold with [Ser(Bzl7)]dermorphin, although it retained a weak mu-selectivity. However, both [Ser(Bzl7)]- and [Ser-NHNHZ7]dermorphin exhibited high affinity for mu receptor sites. Furthermore, the D-configuration about the alpha-carbon of residue 2 and the alpha-amine function and hydroxyl group on Tyr1 are essential for receptor binding. We conclude that mu-opioid receptors contain distinct regions that accomodate the stacked phenol groups of Tyr1 and Tyr5 residues and the phenyl group of Phe3.

Dimeric dermorphin peptides: central administration suppresses gastric acid secretion through interaction with mu-type opioid receptor.

The effect of centrally administered dermorphin and a series of dimeric dermorphin analogs on gastric acid secretion was studied in pylorusligated rats. Dimeric dermorphin analogs consist of identical peptide chains either directly linked with a dihydride bond or a polyethyleneamine bridge at their carboxy termini. At a dose of 0.5 nmol dermorphin, and the dimeric analogs di-[D-Arg2,Sar4]-tetra-DM2, di-penta-DM0, di-[Sar4]-penta-DM0, di-[D-Arg2,Sar4]-penta-DM0, di-[D-Ala4]-penta-DM0 and di-DM0, and di-DM0, inhibited gastric acid output in a statistically significant manner (P less than 0.05). Furthermore, the binding characteristics of these peptides for the mu-type opioid receptor were analyzed using a brain synaptosomal fraction. Dermorphin and several dimeric dermorphin peptides bound to the mu-receptor: di-penta-DM0, di-[Sar4]-penta-DM0, di-penta-DM2 and di-DM0, had 3-to 5-fold greater affinity for the mu-receptor than the specific mu-agonist DAGO. These data indicate that a correlation exists between the central mediated gastric inhibitory effect of DM and several dimeric analogs and their affinity for the mu-type opioid receptor in rat brain.