Ureido group containing cyclic dermorphin(1-7) analogues: synthesis, biology and conformation.

Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.     

Dimeric dermorphin analogues as mu-receptor probes on rat brain membranes. Correlation between central mu-receptor potency and suppression of gastric acid secretion

The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.     

Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues

A new series of analogues of the potent opiate-like peptides dermorphins (mainly tetra- and pentapeptides) were synthesized in order to better evaluate the structure-activity relationships. Relative potencies were referred to dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂), the prototype of this class of frog skin peptides. Peripheral opioid activity (guinea pig ileum and mouse vas deferens) was determined for all the dermorphin analogues. For a selected number of them also central analgesic (hot plate and tail-flick tests) and cataleptic activities were assayed in the rat by intracerebroventricular administration.     

Preparation and Binding Properties of Radioiodinated Analogues of Dermorphin and Deltorphin with High Specificity for the μ- and δ-Opioid Receptors

Abstract: The synthesis, purification, chemical characterization, and binding properties of two ¹²⁵I-labeled analogues of dermorphin and deltorphin-I are described. Native deltorphin-I and [Lys⁷]dermorphin sequences were elongated by an aminopentyl chain on their C-terminal amide function and alkylated with the ¹²⁵I-labeled monoiodinated derivative of Bolton-Hunter reagent (BH^*). The resulting radiolabeled peptides, ε-BH^* [Lys⁷]dermorphin 5-aminopentylamide and ω-BH^* deltorphin-I 5-aminopentylamide, have kept most of the original properties of the parent peptides. They bind with high selectivity and specificity to the μ- (dermorphin analogue) or δ- (deltorphin-I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the μ and δ receptors. The autoradiographic distribution of specific binding sites for the ¹²⁵I-labeled dermorphin and deltorphin-I analogues in rat brain is in complete agreement with previously reported localizations of μ- and δ-opioid receptors. The two radiolabeled peptides are the best ligands of μ- and δ-opioid receptors currently available in terms of sensitivity, specificity, and selectivity.     

New insights on mu/delta selectivity of opioid peptides: conformational analysis of deltorphin analogues

The message domain of dermorphin (Tyr-D-Ala-Phe), a natural mu-opioid heptapeptide, has long been considered the main cause of the high mu selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolor of deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of mu-selective peptides, but endowed of an extremely high delta selectivity, the highest of all natural opioid peptides. A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for mu and delta opioids that recognize the same beta-turn in the N-terminal part, but discriminate for the conformation and polarity of the C-terminal part. Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.     

Cyclic enkephalin and dermorphin analogues containing a carbonyl bridge.

Four cyclic enkephalin analogues and four cyclic dermorphin analogues have been synthesized. Cyclization of linear peptides containing basic amino acid residues of various side chain length in position 2 and 5 (enkephalin analogues) or 2 and 4 (dermorphin analogues) was achieved by treatment with bis-(4-nitrophenyl) carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse activity was observed, depending on the size of the ring and the location of the urea unit. The conformation of two dermorphin analogues has been studied: one of high activity (IC(50) = 4.15 nM in the GPI assay) and a second of low activity (IC(50) = 6700 nM in the GPI assay). The conformational space of these peptides was examined using the EDMC method. Using data from the NMR spectra, each peptide was described as an ensemble of conformers. Biological activity was discussed in light of the structural data.     

Opioid peptides. Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) were replaced by N alpha-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of mu, delta, and kappa opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the kappa binding site. The introduction of N alpha-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed mu/delta specificity.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and activity of dermorphin analogues containing unusual amino acid residues

Summary Solid phase syntheses of analogues of the opioid heptapeptide dermorphin (H-Tyr-dAla-Phe-Gly-Tyr-Pro-Ser-NH₂) containing in the first position 3-aminotyrosine, 3-nitrotyrosine, 4-aminophenylalanine, or nucleoamino acids, 3-(uracilyl-1)alanine, 3-(thyminyl-1)alanine and 3-(6-methyluracilyl-1)alanine are described. The receptor binding properties and analgesic activity of the analogues were examined in comparison with dermorphin. All analogues showed low opioid activity in the binding assays with respect to μ- and δ-receptors. The peptide containing 3-(thyminyl-1)alanine demonstrated a high analgesic activity in different tests when administered intracisternally in mice.     

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Structure-activity studies of dermorphin. The role of side chains of amino acid residues on the biological activity of dermorphin

Seventeen analogues of dermorphin were synthesized and bio-assayed to determine the influence of side chains of the individual amino acid residues forming the sequence of dermorphin on the biological activity of this opioid peptide. Syntheses were carried out using solid-phase procedure, and the analogues obtained were purified by gel filtration on Sephadex G-10. Biological activities determined in guinea pig ileum (GPI) and mouse vas deferens (MVD) tests showed that the N-terminal tetrapeptide is responsible for the activity of dermorphin. Substitutions in the C-terminal fragment, particularly in position 5, for other amino acid residues results in substantial differentiation towards mu and delta receptors.     

Dmt and opioid peptides: a potent alliance

The introduction of the Dmt (2',6'-dimethyl-L-tyrosine)-Tic pharmacophore into the design of opioid ligands produced an extraordinary family of potent delta-opioid receptor antagonists and heralded a new phase in opioid research. First reviewed extensively in 1998, the incorporation of Dmt into a diverse group of opioid molecules stimulated the opioid field leading to the development of unique analogues with remarkable properties. This overview will document the crucial role played by this residue in the proliferation of opioid peptides with high receptor affinity (K(i) equal to or less than 1 nM) and potent bioactivity. The discussion will include the metamorphosis between delta-opioid receptor antagonists to delta-agonists based solely on subtle structural changes at the C-terminal region of the Dmt-Tic pharmacophore as well as their behavior in vivo. Dmt may be considered promiscuous due to the acquisition of potent mu-agonism by dermorphin and endomorphin derivatives as well as by a unique class of opioidmimetics containing two Dmt residues separated by alkyl or pyrazinone linkers. Structural studies on the Dmt-Tic compounds were enhanced tremendously by x-ray diffraction data for three potent and biologically diverse Dmt-Tic opioidmimetics that led to the development of pharmacophores for both delta-opioid receptor agonists and antagonists. Molecular modeling studies of other unique Dmt opioid analogues illuminated structural differences between delta- and mu-receptor ligand interactions. The future of these compounds as therapeutic applications for various medical syndromes including the control of cancer-associated pain is only a matter of time and perseverance.     

6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid mimics active conformation of tyrosine in opioid peptides

6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (6Htc) has been proposed as a rigid mimic of tyrosine conformation in opioid ligand-receptor complex. The significant receptor binding to mu and delta opioid receptors of respective analogues of deltorphin, dermorphin, and endomorphin with D,L-6Htc prove initial prediction.     

Potent side-chain to side-chain cyclized dermorphin analogues containing a carbonyl bridge.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.     

Direct electrophilic fluorination of tyrosine in dermorphin analogues and its effect on biological activity, receptor affinity and selectivity.

In a preliminary communication we reported [(Tetrahedron Lett. 31, 619 (1990)] that acetyl hypofluorite can be used efficiently to introduce fluorine regiospecifically (ortho to OH) into the phenolic ring of tyrosine-containing peptides. This procedure has been applied to the fluorination of a number of mu-selective opioid peptides derived from dermorphin. While the procedure can be used even when the side chains of Arg, Lys, and Tyr are left unprotected, the sulfoxide of a Met(O)-containing analogue was oxidized to sulfone faster than fluorination of the phenolic ring. This method can also be used when the peptide is attached to Merrifield resin. Thus, Tyr(3-F)-D-Ala-Phe-Gly-NH2 and Tyr(3-F)-D-Arg-Phe-Lys-NH2 (F-DALDA) have been prepared, purified, and characterized. Affinities of these fluorinated peptides for both mu- and delta- opioid receptors are reduced (two- to nine-fold) relative to their nonfluorinated analogues, but their selectivity for mu-opioid receptors is not significantly altered. Similarly, the in vitro biological potencies (GPI and MVD assays) of the fluorinated analogues are reduced (two- to seven-fold) relative to their nonfluorinated parent peptides. Thus, F-DALDA, which has high affinity (Ki mu = 15.2 nM) and selectivity (Ki delta/Ki mu = 5390) for mu-opioid receptors, has potential use in biochemical studies which utilize 19F or 18F- labeled compounds.     

Specific opioid binding sites for dermorphin in rat brain. A radioreceptor assay using the tritiated hormone as primary ligand

Dermorphin, a heptapeptide amide isolated from amphibian skin, is the most potent of the naturally occurring opioid peptides. (3H)-dermorphin (52 Ci/mmol, 1294 GBq/mmol) was prepared by catalytic tritiation of the synthetic (2,5-iodotyrosyl 1,5)-dermorphin precursor. High affinity specific binding sites for dermorphin were labeled in rat brain membranes using tritiated dermorphin as primary ligand. The binding was saturable and time-dependent. Scatchard analysis revealed a single population of non-interacting high affinity sites (Kd = 0.86 nM). Dermorphin and the specific opiate antagonist naloxone inhibited specific (3H)-dermorphin binding in a concentration dependent manner. The displacement curves could be fit to a simple competitive model assuming only one population of binding sites, with IC 50 of 1.6 nM and 3.4 nM for dermorphin and naloxone, respectively. The use of tritiated dermorphin will be helpful to ascertain unequivocally the selectivity of dermorphin for the different opioid receptor subtypes in the central nervous system.     

Structural requirements for dermorphin opioid receptor binding

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) was the most potent competitor in all experiments. The D-configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of D-Ala2 with L-Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH2 and dermorphin-(1-3)-NH2, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.     

Characterization of the receptor binding profile of (3H)-dermorphin in the rat brain

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having a D-amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective mu-receptor ligand (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAGO) had similar high potencies in competing with (3H)-dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (D-Pen2, D-Pen5)-enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to mu and delta binding sites by determining its potency in displacing (3H)-DAGO and (3H)-DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DAGO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical mu-receptor probe. The high affinity and selectivity of (3H)-dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the mu-receptor(s).     

Dehydro-dermorphins. II. Synthesis and biological activity of unsaturated dermorphin hexa and heptapeptides

The Phe3 and/or Tyr5 residues in dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and its N-terminal hexapeptide-amide were replaced by delta-Phe or by Phe5 in order to examine the effect on opioid activity. On GPI preparation, the substitution of Phe5 for Tyr5 was well tolerated, whereas the hexa and heptapeptides containing delta Phe in position 3 and/or 5 displayed low potency. The unsaturation at position 3 alone or at positions 3 and 5 was particularly detrimental to mu activity. In the tail flick test, the influence of unsaturation or substitution at positions 3 and 5 generally matched the results of the in vitro assay. Dehydropeptides showed comparatively low antinociceptive effects and [Phe5] analogues displayed about 50% of the analgesic potency of the original peptides.