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.     

Central and peripheral components of dermorphin's effect on rat intestinal propulsion in comparison to morphine

Dermorphin, injected intracerebroventricularly (ICV) to rats, provokes, like to morphine, an inhibition of intestinal propulsion linearly related to the log of the administered doses (in the range from 0.06 to 0.56 μg/rat), but it is 143 times more active than morphine. Naloxone, ICV or IP, antagonizes dermorphin less effectively than morphine. Quaternary naloxone ICV administered antagonizes the intestinal effect of ICV dermorphin, while IP administered it is not effective until 8 mg/kg. The dose of dermorphin maximally active by the ICV route (0.56 μg/rat) is completely inactive when injected IP. Increasing doses of dermorphin IP (from 12 to 6400 μg/kg) inhibit intestinal propulsion to the same extent irrespectively of the doses employed, but never by more than 50%. Only a high dose of naloxone (30 mg/kg/IP) antagonizes this IP effect. The central and peripheral components of this intestinal effect of dermorphin are discussed.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2. The modifications concern two consecutive peptide bonds (Phe3-Cly4-Tyr5, I) or a single one (Gly4-Tyr5-, II or Phe3-Gly4, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2-5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intracerebro or subcutaneous administration.     

Dermorphin: synthesis of analogs and structuro-functional relations

The review analyzes structure-activity relations among dermorphin analogues. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is one of natural opioid peptides having a unique structure and exerting a very potent and prolonged antinociceptive effect. Methods of dermorphin synthesis are summarized together with data on more than 300 dermorphin-like peptides: the physico-chemical characteristics and data on opioid tests in vitro and in vivo are discussed. Based on these studies, conclusions have been drawn on the functional role of each amino acid residue in the dermorphin molecule and on modifications leading to analogues with high and differential opioid activity.     

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).     

The relationship between the structure of dermorphines and their thermoregulatory activity

The effect of structural modifications in the molecule of dermorphin on the functional state of thermoregulation of rats under cold, neutral, and hot environment has been studied. Thermoregulatory and vasomotor activities of 43 peptides (fragments and analogs) were tested in comparison with dermorphin. The role of amino acid residues in the second, third, fourth, and sixth positions was studied. Different fragments of dermorphin molecule proved to underlie different types of thermoregulatory activity (the hypothermic effect was observed in the N-terminal tripeptide, while the N-terminal hexapeptide had the vasodilator activity), and targeted modification of dermorphin molecule makes it possible to separate the hypothermic, vasodilatory, and vasoconstrictive activities. It was proposed that dermorphin is the ligand not only for μ-opioid receptors.     

Synthesis and opioid activity of [Sar4]dermorphin-tetrapeptide analogues

The modification of the dermorphin-(1-4)-tetrapeptide structure led to analogues with potent opioid activity in vitro and in vivo. [Sar4]Tetrapeptides such as H2N-CH(NH)-Tyr-D-Ala-Phe-Sar-D-NH-CH(CH3)C6H5 (VII) whose terminal amino group is replaced by the guanidino function and whose C-terminus is amidated by (R)-(+)-alpha-methylbenzylamine, show peripheral and central opioid activities comparable to or higher than those of dermorphin. The potency of VII in the different tests was as follows: guinea-pig ileum (GPI) IC50 = 0.09nM, mouse Vas deferens (MVD) IC50 = 0.69nM, tail-flick ED50 = 8.91 pmol/mouse, i.c.v. and 4.54 mumol/kg, s.c. This dermorphin-(1-4)-tetrapeptide derivative is about 650 and 950 times as active as morphine in the two in vitro tests, respectively. The MVD/GPI potency ratio of the new peptides suggests a mu-type agonist behaviour.     

Prolactin releasing and luteinizing hormone inhibiting activity of dermorphin shorter homologues in the rat

Dermorphin, a heptapeptide isolated from the skin of the frogs Phillomedusa sauvagei and Phillomedusa rhodei, is endowed with potent peripheral and central opioid-like activity. Intracerebroventricular (icv) injection of dermorphin (31.2, 62.5 and 125 pmol/100g) induced in ovariectomized (OVX) rats dose related rises and decreases in prolactin (PRL) and luteinizing hormone (LH) levels, respectively. The aim of this work was to evaluate the same endocrine responses after administration of shorter peptide amide homologues, related to the N-terminal sequence of dermorphin. These compounds retain a substantial analgesic activity although the latter decreases with the decrease in the number of amino acid residues. Icv administration of the hexapeptide homologue (dermorphin 1-6 amide) to OVX rats did not induce any PRL rise or LH inhibition, even at the high dose of 250 pmol/100g. The pentapeptide (dermorphin 1-5 amide), instead, increased PRL and decreased LH secretion, although the effect was significant only at the dose of 250 pmol/100g. Administration of the tetrapeptide (dermorphin 1-4 amide) induced a significant PRL rise and LH inhibition at both the doses of 125 and 250 pmol/100g. The tetrapeptide was the smallest fragment of the dermorphin moiety which caused endocrine responses while the tripeptide (dermorphin 1-3 amide) was completely ineffective in this context. These data indicate that a complete dissociation exists between the behavioral and endocrine effects of the dermorphin homologues examined. In fact, shorter dermorphins whose analgesic potency was directly related to the number of amino acids, exhibited an opposite pattern in evoking endocrine effects.     

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.     

Dermorphin decreases plasma LH levels in human: evidence for a modulatory role of gonadal steroids

The purpose of this study was to evaluate the effects of dermorphin, a new synthetic powerful opiate-like heptapeptide, on plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in fertile and postmenopausal women. In fertile subjects, dermorphin (5.5 micrograms/kg min for 30 min) decreases plasma LH (p less than 0.01 vs. baseline and placebo values), but not plasma FSH. The area under the curve during dermorphin infusion was significantly lower than during placebo infusion (p less than 0.01). Pretreatment with the opioid receptor antagonist naloxone, blocked the decrease of plasma LH levels. In postmenopausal women not subjected to any treatment, dermorphin infusion did not significantly modify plasma LH and FSH levels. On the contrary, its administration to postmenopausal subjects treated with conjugated estrogens and medroxyprogesterone acetate significantly decreased plasma LH levels (p less than 0.01, vs. baseline, placebo and area under the curve). Considering the modulatory role exerted by ovarian steroids on the activity of such receptors, these data also indicate that opioid systems play a very important part in the hypothalamus-pituitary-ovarian axis.     

Effects of corticotropin-releasing factor (CRF), tuftsin and dermorphin on behavior of squirrel monkeys maintained by different events

The effects of intracerebroventricular (ICV) administration of ovine CRF (0.1–30.0 μg/kg), dermorphin (0.3–30.0 μg/kg) and tuftsin (10–3000 μg/kg) were examined in squirrel monkeys trained to respond under a multiple 3-min fixed-interval schedule of food presentation and either shock presentation or stimulus-shock termination. Initial administration of the 41-amino acid polypeptide CRF increased food-maintained responding by 150–200% in 2 of 3 subjects. However, no other doses tested affected response rates, a result that may have been due to the rapid development of tolerance. The tetrapeptide tuftsin selectively increased responding maintained by food presentation at doses that decreased shock-maintained responding. The heptapeptide dermorphin selectively increased food-maintained responding when responding in the other component of the multiple schedule was maintained by shock presentation. When responding was maintained by a multiple food, stimulus-shock termination schedule, dermorphin decreased response rates in both components. Dermorphin's rate increases were blocked by the opiate antagonist naloxone, indicating that dermorphin's actions were mediated through the opiate receptor. These results indicate that the behavioral effects of tuftsin, dermorphin, and perhaps CRF, depend on the manner in which responding is controlled by its consequences. While the actions of tuftsin and dermorphin are believed to be mediated through the opiate system, the behavioral effects observed in primates appear different from the effects of morphine under similar schedule conditions.     

Effects of Aging and Morphine Administration on Calmodulin and Calmodulin-Regulated Enzymes in Striata of Mice

Male ICR mice, young (25-days old), mature (3-months old), and old (22 months), were injected with morphine sulfate (10 mg/kg, s.c.) or were implanted with morphine pellets (75 mg). Controls received saline injections or placebo pellets. One hour after injections and 72 h after pellet implantations, the mice were decapitated and striatal regions were removed for the following analyses: calmodulin (CaM) levels via radioimmunoassay and activities of cyclic nucleotide phosphodiesterases, adenylate and guanylate cyclases, and Ca2+, Mg2+-ATPase. Acute morphine treatment produced the following: (1) increases in calmodulin levels in the young and old mice while having no effect on mature levels; (2) increases in activities of guanylate cyclase of mature mice while decreasing those of the old mice; (3) no effects on activity of adenylate cyclase; (4) decreased activity of cyclic AMP-phosphodiesterase in young mice only; (5) decreased activity of Ca2+, Mg2+-ATPase in the old mice only. The only changes found in striata from morphine-tolerant mice when compared with age-matched controls were elevations in cyclic GMP-phosphodiesterase activities in all three age groups. Differences in control values of the three age groups were as follows: CaM levels, mature greater than old greater than young; Ca2+, Mg2+-ATPase activity, old greater than mature-young. The results indicate age-induced changes in cellular regulation and biochemical responses to morphine.     

Synthesis and biological studies of dermorphin and its analogs substituted at positions 5 and 7

Dermorphin and seven of its analogs substituted at positions 5 and/or 7, have been synthesized by the solid phase method employing mainly 9-fluorenylmethyloxycarbonylamino acid trichlorophenyl esters in presence of l-hydroxybenzotriazole, the solid support being the Merrifield resin. Among the analogs synthesized, the most interesting is [Tyr7]dermorphin. It is one of the most potent dermorphin analogs reported so far. Compared to the natural peptide, it is about two times more potent in the GPI (in vitro) and nearly 1.4 times more potent in its analgesic activity in mice by the hot plate test (in vivo). Further, its antidiarrhoeal activity in mice (in vivo) is comparable to that of dermorphin. On the other hand, [Thr7]dermorphin is almost as potent as dermorphin.     

Opioid activity of delta O-endorphin in the guinea pig ileum.

The oligopeptides beta- and delta O-endorphin were isolated from porcine and bovine pituitary respectively. Their opiate activity was determined in the guinea pig ileum and compared to that of the pentapeptide methionine-enkephalin and morphine. The rank order of opioid activity was found to be: morphine greater than beta-endorphin = Met-enkephalin greater than delta O-Endorphin which lacks the four C-terminal amino acids of beta-endorphin displayed 60% of the activity of beta-endorphin. These results indicate, that C-terminal amino acids contribute little to the affinity of beta-endorphin for opiate receptors in the guinea pig ileum.     

Effect of Structural Changes of Pro6 in Dermorphin Molecule on Its Antinociceptive Activity

We studied effect of dermorphin (H-Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH₂) and its analogs with modified amino acid residue proline in position 6, H-Tyr-DAla-Phe-Gly-Tyr-[DPro]-Ser-NH₂, H-Tyr-DAla-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH₂, and H-Tyr-DAla-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH₂, on nociception in the tail-flick and hot plate tests after intraperitoneal injection. Replacement of LPro with the stereoisomer DPro as well as Pro dehydration (LdHPro) was shown to increase antinociceptive activity. Replacement of LdHPro with DdHPro cancelled the activity in the tail-flick test. All three dermorphin analogs retained antinociceptive activity in the hot plate test; however, the effect of dermorphin was more pronounced.     

Paradoxical correlations of the effectiveness of the intranasal and intraperitoneal administration of dermorphins in rats

The analgetic activity of dermorphin and its analogue A-2 was assessed by the tail-flick test. Following intraperitoneal administration at doses 5 mg/kg and above the peptides showed significant effects. After intranasal application of the peptides a significant effect was observed in the range of low doses 0.001-0.1 mg/kg. After intranasal application of high dermorphin doses (1 or 5 mg/kg) the analgetic activity decreased. The effect of analogue A-2 lasted longer after intranasal, than after intraperitoneal administration. It is assumed that the neurophysiological mechanisms of the analgetic activity of dermorphins depend on the route of their administration.     

The potential-dependent action of the opioid peptide dermorphin]

Dermorphin action was studied on cross-section strip of frog stomach muscle by a mechanographic recorder. The results show that dermorphin (10(-5)-10(-8) M) blocks acetylcholine effects, spontaneous activity and muscle contractions induced by direct electrical stimulation. All the above effects are hardly reversible. Dermorphin fails to block spontaneous and evoked activities if it is injected into the incubated medium during K(+)-depolarization (KCI--100 mM) of the muscle. Thus, dermorphin has voltage-dependent action. The discussion deals with dermorphin action on voltage-dependent Ca(2+)-channels of muscle cell membrane.     

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.