Response of growth hormone release to human growth hormone-releasing factor and its analogs in the bovine

Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH2 analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH2 in female dairy calves. Four- and 12-month-old calves were injected intravenously with 0.25 microgram of hGRF-44-NH2 or its analogs per kg of bw. Blood samples were collected before, and during 180 min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180 min after injection of hGRF-44-NH2 and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF(1-26)-NH2 was significantly lower than that of hGRF-44-NH2 (P less than 0.05). On the other hand, hGRF(1-29)-NH2 possessed similar potency to hGRF-44-NH2. [D-Tyr1]-hGRF-44-NH2 showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH2. Also, [D-Ala2]-hGRF(1-29)-NH2 exhibited prolonged GH-releasing activity, and its potency was 2.5 (P less than 0.05) and twice (P less than 0.05) as great as that of hGRF-44-NH2 and hGRF(1-29)-NH2, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala2]-hGRF(1-29)-NH2 has longer and greater activity, on a dose basis, than hGRF-44-NH2 in the calves.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-inflammatory activity of alpha-MSH(11-13) analogs: influences of alteration in stereochemistry.

D-Amino acid substitutions in the anti-inflammatory/antipyretic Ac-alpha-MSH(11-13)-NH2 tripeptide of Ac-alpha-MSH(1-13)-NH2 were made and the altered peptides were injected in mice treated with picryl chloride. Ear swelling, measured 3 and 6 h after application of the irritant, was reduced by IP injections of Ac-alpha-MSH(11-13)-NH2, in confirmation of previous observations. Ac-[D-Lys11]alpha-MSH(11-13)-NH2 effected similar anti-inflammatory activity but Ac-[D-Pro12]alpha-MSH(11-13)-NH2 was inactive. Ac-[D-Val13]alpha-MSH(11-13)-NH2 and Ac-[D-Lys11,D-Val13]alpha-MSH(11-13)-NH2 generally had greater anti-inflammatory activity than the parent tripeptide molecule; the dose-response relations exhibited the bell-shaped characteristics seen previously with MSH peptides. The results indicate that the L-Pro12 is essential for the anti-inflammatory activity of Ac-alpha-MSH(11-13)-NH2 whereas the L-Lys11 is not. D-Val13 substitution increased anti-inflammatory activity approximately four-fold over Ac-alpha-MSH(11-13)-NH2. These results provide new structure-activity relationships of the anti-inflammatory Ac-alpha-MSH(11-13)-NH2 molecule. The data support the developing idea that alpha-MSH and its COOH-terminal fragments modulate host responses, perhaps by antagonizing the actions of cytokines.     

The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.     

Alpha-melanocyte stimulating hormone message and inhibitory sequences: comparative structure-activity studies on melanocytes

We investigated the structure-activity relationships of alpha-MSH (alpha-melanocyte stimulating hormone) fragment derivatives of the generic formulae Ac-alpha-MSH(x-13)-NH2 and Ac-alpha-MSH(6-x)-NH2. The minimal C-terminal sequences required for melanotropic activity were 8-13 and 7-13, respectively, in the frog and lizard skin bioassays. The Arg8-Trp9 sequence appears to be a fundamental component of the minimal message sequences found to date such as alpha-MSH(6-9), alpha-MSH(8-13) and alpha-MSH(7-13). We discovered that Ac-alpha-MSH(7-10)-NH2 was a weak and selective alpha-MSH antagonist on the lizard skin bioassay. Analysis of alpha-MSH(7-10) analogues of the generic formula Ac-Xaa-Arg-Trp-Yaa-NH2 led to Ac-[D-Trp7,D-Phe10]alpha-MSH(7-10)-NH2, a moderately potent, specific and competitive inhibitor of alpha-MSH in both the frog and the lizard skin bioassays.     

Nociceptin, OP4 receptor ligand in different models of experimental epilepsy

The anticonvulsive activity of nociceptin, endogenous OP4 receptors agonist was investigated in pentylenetetrazole (PTZ), N-methyl D-aspartic acid (NMDA), bicucculine (BCC) and electrically evoked seizure models of experimental epilepsy. Nociceptin, at the dose of 10 nmol, suppressed the clonic seizures induced by PTZ, NMDA and BCC. [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which has been proposed to be selective antagonist OP4 receptors, did not prevent the action of nociceptin. The effect of [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 on seizures induced by PTZ, NMDA and BCC was very similar to that of nociceptin. These data support the hypothesis that it possesses agonistic properties. Naloxone did not reverse the anticonvulsive action of nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which excludes the participation of opioid receptor in this action. On the other hand in the electroconvulsive model of generalized seizures, nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 influenced neither the electroconvulsive threshold nor the maximal electroshock test. The data suggest that nociceptin and [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 can exert anticonvulsive action. These properties depend on OP4 but not opioid receptors activation.     

Cyclic melanotropins. Part VII: Modified ring structures--synthesis and biological activity

The highly potent cyclic analogue of alpha-MSH, Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa4,Cys10]-alpha-MSH4-13-NH2 resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys4,Cys10[-alpha-MSH4-a3-NH2 led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa2,Cys10]-alpha-MSH4-13-NH2, resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the righ by methylene group to give Ac-[Hcy4,Cys10]-alpha-MSH4-13-NH2 led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.     

Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.     

Low-molecular-weight anti-HIV-1 peptides from the amino-terminal sequence of RANTES: possible lead compounds for coreceptor-directed anti-HIV-1 agents.

A series of small peptides corresponding to the amino-terminal sequence of RANTES were synthesized, and their anti-HIV-1 activity was evaluated. Pentapeptides, H-(10Ala-RANTES 6-10)-OH and Ac-(10Ala-RANTES 6-10)-NH2, were the smallest anti-HIV-1 peptides so far developed, and would be potentially important lead compounds for coreceptor-directed anti-HIV-1 drugs.     

Degradation of aspartic acid and asparagine residues in human growth hormone-releasing factor.

Products of the degradation of human growth hormone-releasing factor (GRF) in aqueous solutions (15-200 microM) have been isolated and fully characterized. The cleavage product, GRF(4-44)-NH2, and the isomerization product, [beta-Asp3]GRF(1-44)-NH2, from the degradation of GRF(1-44)-NH2 in acidic solution and the corresponding products, GRF(4-29)-NH2 and [beta-Asp3]GRF(1-29)-NH2, from the degradation of GRF(1-29)-NH2 have been isolated and characterized. The products, [beta-Asp8]GRF(1-44)-NH2 and [Asp8]GRF(1-44)-NH2, from the deamidation of GRF(1-44)-NH2 at pH 8.0 and the corresponding products, [beta-Asp8]GRF(1-29)-NH2 and [Asp8]GRF(1-29)-NH2, from the deamidation of GRF(1-29)-NH2 have been isolated and characterized. All the degradation products of GRF(1-44)-NH2 and GRF(1-29)-NH2 were evaluated for biological activity and found to have much lower in vitro potencies than the parent peptides. Degradation occurs at Asp3 and Asn8 and the kinetics of these various transformations versus pH and temperature have been studied. GRF is most stable at pH 4-5. At pH below the pKa of the Asp3 side-chain (pH less than 4), cleavage at Asp3-Ala4 is the major route of degradation. For pH greater than 4, isomerization of Asp3 to beta-Asp3 (iso-Asp3) predominates. The rates of cleavage and isomerization are simple first order and vary with pH, independent of buffer concentration, such that the protonated (COOH) form of Asp3 undergoes cleavage while the ionized (COO-) form isomerizes. The more rapid deamidation of Asn8 to generate beta-Asp8 and Asp8 in about a 4:1 ratio, presumably via a cyclic imide intermediate, occurs at pH greater than or equal to 5 and is general base-catalyzed. Evidence was also obtained for direct hydrolysis of protonated Asn8 in GRF(1-29)-NH2 at pH less than or equal to 2 to give exclusively [Asp8]GRF(1-29)-NH2. The deamidation of Asn8 in GRF(1-29)-NH2 at pH 8.0, 22-55 degrees C, is relatively insensitive to temperature for T less than 37 degrees C, possibly due to conformational constraints. Asp25 and Asn35 are sterically, conformationally, or otherwise hindered with respect to these changes as no degradation at these sites was observed under the conditions employed.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

Receptor binding affinity and thermolysin degradation of truncated and retro-inverso-isomeric ANF analogs

The peptides H-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-NH2 (rANF8-15-NH2), Ac-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-NH2 (Ac-rANF8-15-NH2), and their corresponding retro-inverso-isomeric peptides H-D-Ile-D-Arg-D-Asp-D-Ile-D-Arg-Gly-Gly-D-Phe-NH2 (D-rANF15-8-NH2), Ac-D-Ile-D-Arg-D-Asp-D-Ile-D-Arg-Gly-Gly-D-Phe-NH2 (Ac-D-rANF15-8-NH2), were evaluated for their ability to compete for the binding of 125I-rANF5-28 to cultured spontaneously hypertensive rat (SHR) aortic smooth muscle cell membranes. Their stability toward hydrolysis by the neutral endopeptidase thermolysin was also studied. The octapeptides rANF8-15-NH2 and Ac-rANF8-15-NH2 bound with IC50's of 367 pM and 1900 pM, respectively, but were rapidly hydrolyzed by thermolysin. Retro-inverso-isomers were prepared to provide molecules with an improved enzymatic stability. The retro-inverso-isomers were completely stable to thermolysin but were virtually inactive in the binding assay (IC50 greater than 1 microM).     

Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.     

Action of peptidases in brain synaptic membranes on the NH2-terminus of adrenocorticotropin using ACTH-(1-16)-NH2 as a model substrate

The action of brain peptidases on NH2-terminal sequences of adrenocorticotropin was studied by incubation of ACTH-(1-16)-NH2 under different pH conditions. Profiles of metabolites and time course of product formation were obtained by HPLC analysis of the digests. Fragments of ACTH-(1-16)-NH2 were isolated and characterized by their amino acid composition and NH2-terminal groups. Both at pH 7.4 and pH 8.5 the following fragments were found: ACTH-(3-16)-NH2, ACTH-(4-16)-NH2, ACTH-(5-16)-NH2, and ACTH-(7-16)-NH2. At pH 7.4 the major products were ACTH-(4-16)-NH2 and ACTH-(7-16)-NH2, while the peptide ACTH-(3-16)-NH2 was the main metabolite at pH 8.5. The nature of identified peptides and the time course of their formation demonstrates that aminopeptidase activities predominate in the conversion of the NH2-terminus of adreno-corticotropin and related peptides by brain synaptic membranes.     

Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor referred to as N/OFQ peptide (NOP) receptor. NOP receptor activation by N/OFQ modulates several biological functions both at central and peripheral level. Structure activity relationship (SAR) studies demonstrated that the N/OFQ sequence can be divided into a N-terminal tetrapeptide 'message' crucial for receptor activation and a C-terminal 'address' important for receptor binding. On the basis of this message/address concept we synthesized some chimeric compounds in which we substituted the natural message domain with the nonselective nonpeptide NOP ligand (8-Naphthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-aceticacid methyl ester (NNC 63-0532) and used as address domain the peptide sequences Thr-NH2, N/OFQ(5-9)-NH2, N/OFQ(5-13)-NH2 and N/OFQ(5-17)-NH2. All the compounds were pharmacologically evaluated in the electrically stimulated guinea-pig ileum. NNC 63-0532 produced a concentration-dependent inhibition of the electrically induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the NOP selective antagonist [Nphe1, Arg14, Lys15]N/OFQ-NH2 (UFP-101) while prevented by naloxone. Similar results were obtained with NNC 63-0532/Thr-NH2 and NNC 63-0532/N/OFQ(1-9)-NH2. On the contrary, the inhibitory effects of NNC 63-0532/N/OFQ(5-13)-NH2 and NNC 63-0532/N/OFQ(5-17)-NH2 were slightly antagonized by UFP-101 while naloxone prevented the effects of the high but not of the low concentrations of the two ligands. These data indicate that it is possible to functionalize with the N/OFQ address sequence a nonpeptide NOP ligand for increasing its binding to the NOP receptor. Moreover, these results corroborate the idea that the 5-13 sequence represents the crucial core of the N/OFQ address domain.     

Analysis of the interaction between lectins and tetra- and tri-saccharide mimics of the Sd(a) determinant by surface plasmon resonance detection

The binding properties of a spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (1), two tetrasaccharide mimics beta-D-Galp-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (2) and beta-D-GlcpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (3), and two trisaccharide mimics beta-D-GalpNAc-(1-->4)-3-O-(SO(3)H)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (4) and beta-D-GalpNAc-(1-->4)-3-O-(CH(2)COOH)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (5) with lectins from Dolichos biflorus (DBL), Maackia amurensis (MAL), Phaseolus limensis (PLL), Ptilota plumosa (PPL), Ricinus communis 120 (RCL120) and Triticum vulgaris (wheat germ agglutinin, WGA) have been investigated by surface plasmon resonance (SPR) detection. MAL, PPL, RCL120 and WGA did not display any binding activity with compounds 1-5. However, DBL and PLL, both exhibiting GalNAc-specificity, showed strong binding activity with compounds 1, 4 and 5, and 1, 3, 4 and 5, respectively. The results demonstrate that SPR is a very useful analysis system for identifying biologically relevant oligosaccharide mimics of the Sd(a) determinant.