deltorphin and dermorphin

In 1980 the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists, and named dermorphins. Since 1987 deltorphins, a family of highly selective delta-opioid peptides were identified either by cloning of the cDNA from frog skins or isolation of the peptides. The distinctive feature of opioid peptides is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe. The discovery of the amphibian opiate peptides, provided new insights into the functional role of the mu- and delta-opiate systems. It also provided models for novel analgesics with enhanced therapeutic benefits and reduced toxicity.

Topics: Animals; Bufonidae; DNA, Complementary; Oligopeptides; Opioid Peptides; Peptides; Receptors, Opioid, delta; Receptors, Opioid, mu; Skin Physiological Phenomena; Structure-Activity Relationship

Naturally occurring environmental substances often mimic endogenous substances found in mammals and are capable of interacting with specific proteins, such as receptors, with a high degree of fidelity and selectivity. Narcotic alkaloids and amphibian skin secretions, introduced into human society through close association with plants and animals through folk medicine and religious divination practices, were incorporated into the armamentarium of the early pharmacopoeia. These skin secretions contain a myriad of potent bioactive substances, including alkaloids, biogenic amines, peptides, enzymes, mucus, and toxins (noxious compounds notwithstanding); each class exhibits a broad range of characteristic properties. One specific group of peptides, the opioids, containing the dermorphins (dermal morphinelike substances) and the deltorphins (delta-selective opioids), display remarkable analgesic properties and include an amino acid with the rare (in a mammalian context) D-enantiomer in lieu of the normal L-isomer. Synthesis of numerous stereospecific analogues and conformational analyses of these peptides provided essential insights into the tertiary composition and microenvironment of the receptor "pocket" and the optimal interactions between receptor and ligand that trigger a biological response; new advances in the synthesis and receptor-binding properties of the deltorphins are discussed in detail. These receptor-specific opioid peptides act as more than mimics of endogenous opioids: their high selectivity for either the mu or delta receptor makes them formidable environmentally derived agents in the search for new antagonists for treating opiate addiction and in the treatment of a wide variety of human disorders.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Models, Molecular; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Ranidae; Skin

Topics: Amino Acid Sequence; Amino Acids; Amphibians; Animals; Molecular Sequence Data; Mollusca; Oligopeptides; Opioid Peptides; Peptide Biosynthesis; Peptides; Skin; Snails; Stereoisomerism

Topics: Amino Acid Sequence; Analgesics; Analgesics, Opioid; Animals; Endorphins; Humans; Molecular Sequence Data; Oligopeptides; Opioid Peptides

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. The insensitivity of the delta OR (DOR) to deltorphin was due to variation of a single amino acid, Trp7.35, which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish, and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides.

Topics: Amino Acid Sequence; Amphibians; Analgesics, Opioid; Animals; Behavior, Animal; Binding Sites; Humans; Kinetics; Molecular Dynamics Simulation; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Peptides; Protein Structure, Tertiary; Receptors, Opioid; Sequence Alignment; Skin; Species Specificity; Zebrafish

A series of novel cyclic ureidopeptides, analogues of dermorphine/deltorphine tetrapeptide, were synthesized by solid phase peptide synthesis and/or in solution. The antinociceptive activity of N-substituted amides 1-10 was evaluated using hot-plate and tail-flick tests. Analogue 1 showed significant, stronger than morphine, antinociceptive effect after systemic applications. All analogues were also tested for their in vitro resistance to proteolysis by means of mass spectroscopy and it was found that all substituted amides 1-10 showed full stability during incubation with large excess of chymotrypsin and pepsin. Compound 1 is a lead molecule for further evaluation.

Topics: Analgesics, Opioid; Animals; Chymotrypsin; Hot Temperature; Hydrolysis; Hyperalgesia; Indoles; Male; Mice; Mice, Inbred BALB C; Models, Chemical; Molecular Structure; Oligopeptides; Opioid Peptides; Pepsin A; Proteolysis; Spectrometry, Mass, Electrospray Ionization; Styrenes

Topics: Magnetic Resonance Spectroscopy; Oligopeptides; Opioid Peptides; Peptide Fragments; Receptors, Opioid, delta; Receptors, Opioid, mu; Structure-Activity Relationship

The N-terminal tetrapeptide segments of dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and deltorphin (Tyr-D-Ala-Phe-Asp/Glu-Val-Val-Gly-NH(2)) are agonists at the opioid receptors micro and delta, respectively. [D-Arg(2), Lys(4)]-dermorphin-(1-4) amide (Tyr-D-Arg-Phe-Lys-NH(2), DALDA) and [Dmt(1)]DALDA (where Dmt is 2',6'-dimethyltyrosine) are among the most potent and selective micro-agonists reported to date, both in vitro (having picomolar micro receptor affinity) and in vivo. In this communication, conformation-activity studies of the following four cyclic analogs of DALDA are presented and discussed: the lead peptide S(2),S(4)-cyclo (Tyr-D-Cys-Phe-Cys-NH(2)), constrained by means of an S(4.2)--S(4.4) disulfide between Cys(2) and Cys(4); its two cis and trans C(4.2)--C(4.4)-olefinic dicarba analogs, and the product of saturation of them both. They are potent nonselective or moderately micro-selective opioid agonists in vitro.They have been synthesized and tested earlier [Berezowska I, Chung NN, Lemieux C, Wilkes BC, and Schiller PW, Acta Biochim Polon 53, 2006, 73-76]. We have studied their conformations using NMR and molecular dynamics. With major conformational constraints imposed by the 11-membered ring spanning residues 2-4, they show well defined conformations of this ring, while the exocylic Tyr(1) and Phe(3) side chains still have significant conformational freedom. The more active and selective micro versus delta disulfide and saturated dicarba agonists seem to have in common: (i) their ring structures more flexible than those of the other two and (ii) their ring structures similar to each other and more diverse than those in the other two. Given this and the small size of the peptides having confirmed bioactivity profiles, there is a chance that their conformations determined in solution approach receptor-bound conformations.

Topics: Computer Simulation; Cyclization; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Oligopeptides; Opioid Peptides; Protein Conformation; Receptors, Opioid, delta; Structure-Activity Relationship

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.

Topics: Amino Acids, Aromatic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain Measurement; Phenylalanine; Receptors, Opioid, mu; Vas Deferens

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.

Topics: Analgesics, Opioid; Animals; Guinea Pigs; In Vitro Techniques; Magnetic Resonance Spectroscopy; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Peptides, Cyclic; Protein Conformation; Receptors, Opioid, delta; Receptors, Opioid, mu

The usefulness of 2,6-dimethylphenylalanine (Dmp) as a Phe surrogate in two opioid peptides, dermorphin (DM) and deltorphin II (DT), was investigated. Compared to DM, [L-Dmp(3)]DM (1) showed a 170-fold increase in mu affinity and only a 4-fold increase in delta affinity, resulting in a 40-fold improvement in mu receptor selectivity. Compared to DT, [L-Dmp(3)]DT (3) showed a 22-fold increase in delta affinity and somewhat of a loss in mu affinity, and consequently a marked (75-fold) improvement in delta receptor selectivity. The D-Dmp replacement, however, resulted in a great loss in receptor selectivity in each of the peptides. The specific receptor interactions of 1 and 3 were confirmed by in vitro bioassays. Analogues 1 and 3 seem to be useful as pharmacological tools for the study of opioid systems.

Topics: Analgesics, Opioid; Animals; Guinea Pigs; Inhibitory Concentration 50; Muscle Contraction; Muscle, Smooth; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Phenylalanine; Protein Binding; Receptors, Opioid; Structure-Activity Relationship

Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 microg of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin.

Topics: Alanine; Animals; Chromatography, Affinity; Immune Sera; Immunoglobulin G; Immunohistochemistry; Magnetic Resonance Spectroscopy; Oligopeptides; Opioid Peptides; Peptide Biosynthesis; Peptides; Phenylalanine; Polylysine; Protein Binding; Rabbits; Ranidae; Skin; Tyrosine

In the present paper we describe the synthesis of some dermorphin and deltorphin analogues beta-O- and alpha-C-glycosylated on the C-terminal amino acid residue and report their opioid receptor affinity and selectivity as well as their analgesic potency after subcutaneous injection in mice.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Glycosylation; Guinea Pigs; In Vitro Techniques; Injections, Subcutaneous; Male; Mice; Muscle, Smooth; Oligopeptides; Opioid Peptides; Receptors, Opioid

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, epsilon-BH* [Lys7] dermorphin 5-aminopentylamide and omega-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 mu- (dermorphin analogue) or delta- (deltorphin-I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the mu and delta 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 mu- and delta-opioid receptors. The two radiolabeled peptides are the best ligands of mu- and delta-opioid receptors currently available in terms of sensitivity, specificity, and selectivity.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Autoradiography; Binding, Competitive; Brain Chemistry; Iodine Radioisotopes; Ligands; Oligopeptides; Opioid Peptides; Organ Culture Techniques; Peptide Fragments; Protein Precursors; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Sensitivity and Specificity

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glycopeptides; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Jejunum; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Structure-Activity Relationship; Synaptosomes

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Topics: Amino Acid Isomerases; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Binding Sites; Kinetics; Molecular Sequence Data; Mollusca; Oligopeptides; Opioid Peptides; Protein Processing, Post-Translational; Ranidae; Ribosomes; Serine; Skin; Substrate Specificity

Opioid peptides serve as tonically active negative growth factors in neural and non-neural cells, in addition to being neuromodulators. To investigate the involvement of opioids in homeostatic renewal of epithelial cells in the epidermis, mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX) (20 mg/kg). Disruption of opioid-receptor interaction by NTX resulted in an elevation of 42 and 72% in DNA synthesis in skin from the dorsum and plantar surface of the hindfoot, respectively, within 2 h; response to NTX was dependent on the circadian rhythm in each region examined. Injection of the naturally occurring and potent opioid growth factor (OGF), [Met5]-enkephalin, at 1 mg/kg depressed DNA synthesis in the dorsum and plantar surface by 42 and 19%, respectively, within 2 h; the effects of OGF complied with the pattern of circadian rhythm in each area of skin. The decreases in labeling index evoked by OGF were blocked by concomitant administration of the opioid antagonist, naloxone (10 mg/kg); naloxone alone at the dosage utilized had no influence on cell replicative processes. In tissue culture studies, OGF and NTX respectively depressed and elevated DNA synthesis. Both OGF and its receptor, zeta, were detected in all but the cornified layer of the epidermis in murine skin from the dorsum, plantar surface, pinnae, and tail. In addition, both peptide and receptor were observed in basal and suprabasal cells of the human epidermis. These results lead to the suggestion that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the skin in a direct manner, regulating DNA synthesis in a tonically inhibitory, circadian rhythm-dependent fashion.

Topics: Animals; Circadian Rhythm; Culture Techniques; DNA; Enkephalin, Methionine; Epidermis; Growth Substances; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Naltrexone; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Receptors, Opioid; Skin

The side chain of Tyr and Phe was fixed into the gauche(-) or gauche(+) conformation by using the Tic Htc structures, and into the trans conformation by using an aminobenzazepine-type (Aba) structure. When incorporated into dermorphin or deltorphin II, the Tic and Htc analogues all showed a large decrease in both mu and delta affinities and activities. Fixation of Phe(3) in the trans rotamer resulted in a large increase in delta affinity in the dermorphin analogue, whereas in the [Aba(3)-Gly(4)] deltorphin II analogue, good delta affinity is maintained despite the removal of the Glu side chain. Whereas several authors propose a gauche(-) preferred conformation for the Phe(3) side chain, these results suggest a trans conformation at the delta receptor. The use of these conformationally constrained residues for evaluating the preferred solution conformation in the flexible N-terminal tripeptide Tyr-D-Ala-Phe is illustrated. The (1)H-nmr parameters--chemical shift, temperature dependence, and nuclear Overhauser effects to the D-Ala(2) methyl protons in the different analogues--provide direct evidence to confirm the proposed sandwich conformation in the native peptides.

Topics: Amino Acid Sequence; Analgesics, Opioid; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Oligopeptides; Opioid Peptides; Phenylalanine; Protein Binding; Protein Conformation; Receptors, Opioid; Stereoisomerism; Tyrosine

To study the effect of the sulfate ester moiety on the opioid activity, we prepared two tyrosine sulfate-(Tyr(SO3H)-containing dermorphin and deltorphin peptides. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membranes and in two bioassays, using guinea pig ileum and mouse vas deferens. In comparison to original peptides and morphine, the obtained data indicate that whereas H-Tyr(SO3H)-D-Ala-Phe-Gly-NH2 shows very minimal interaction with opioid receptors, H-Tyr(SO3H)-D-Ala-Phe-Asp-Val-Val-Gly-NH2 retains a significant activity.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Binding, Competitive; Chromatography, High Pressure Liquid; Guinea Pigs; In Vitro Techniques; Male; Mice; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Rats; Rats, Wistar; Receptors, Opioid; Trifluoroacetic Acid; Tyrosine

The availability of the most selective, high-affinity, natural opioid agonists for mu-receptors (dermorphin-DM) and delta-receptors (deltorphin-DT) has provided the possibility for in vivo studying of the role of acute and chronic activation of mu- and delta-opioid receptors on the functional activity of the hypothalamus-pituitary-adrenocortical (HPA) axis, both in basal conditions and in response to an acute stress in adult male rats. Plasma corticosterone (CS) and beta-endorphin-like-immunoreactivity (beta-EP-LI) levels were measured by specific radioimmunoassays before and after 5 and 30 minutes from the exposure to cold (3 +/- 0.5 C) water and forcing them to swim for 10 minutes (acute cold swimming stress). Acute administration of DM, the specific mu-receptor agonist, enhanced basal and stress induced plasma levels of CS and beta-EP-LI. These effects were antagonized by pretreatment with naloxone, specific mu-opioid receptor antagonist, but not by naltrindole, a delta-opioid receptor antagonist. Long-term administration of DM did not alter resting plasma levels of CS and beta-EP-LI, but significantly reduced stress-induced increase of these hormones. Both the acute and chronic administration of the DT, highly selective delta-opioid receptors agonist, failed to modify resting and stress induced hormone levels. Our present data show that DM throughout mu-opioid receptors, but not DT, modulates the response of HPA axis to acute stress in rats, increasing or decreasing the release of CS and beta-EP-LI when acutely or chronically administered, respectively.

Topics: Adrenal Glands; Amino Acid Sequence; Animals; beta-Endorphin; Cold Temperature; Corticosterone; Hypothalamus; Male; Molecular Sequence Data; Naloxone; Oligopeptides; Opioid Peptides; Pituitary Gland; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Stress, Physiological; Swimming

The N-terminal dipeptide Tyr-d-Ala of a mu-selective agonist, dermorphin tetrapeptide (DT, H-Tyr-D-Ala-Phe-Gly-NH2) and delta-selective agonist deltorphin C (DEL-C, H-Tyr-D-Ala-Phe-Asp-Val-Val- Gly-NH2) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2-bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra- and heptapeptides I-VI were characterized for mu- and delta-opioid receptor binding properties using [3H]DAGO and [3H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N-terminal depeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to mu- and delta-prototypes (500-fold on the mu-site, analog I, and 125-fold on the delta-site, analog IV). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA2 values of 6.05 and 6.82. Analog IV did not exhibit the delta-antagonist potency and delta-selectivity of TIPP peptides.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Guinea Pigs; Ileum; Male; Mice; Molecular Sequence Data; Molecular Structure; Morphine; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Receptors, Opioid; Stereoisomerism; Tetrahydroisoquinolines; Vas Deferens

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Base Sequence; Cloning, Molecular; DNA; Endorphins; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Peptides; Protein Precursors; Vertebrates

In an effort to explore structural features affecting receptor recognition in a series of conformationally restricted tetrapeptides related to the cyclic, delta opioid receptor-selective analogue, [formula: see text] electronic, lipophilic, and steric effects at the Phe3 residue were assessed by substitution at different positions of the side-chain aromatic ring by halogens, alkyl, hydroxyl, and nitro groups. Effects on opioid receptor binding affinity and selectivity were determined. The results, which are generally consistent with reports of analogous modifications in linear and cyclic pentapeptide enkephalins, indicate that steric, lipophilic, and electronic properties are all important determinants of delta opioid receptor recognition. Specifically, modifications which increase lipophilicity or exert electron-withdrawing effects on the aromatic ring enhance binding affinity, while hydrophilic, bulky, or electron-releasing modifications are detrimental. These observations are in excellent agreement with quantitative structure-activity relationship (QSAR) results reported for Phe4 modifications in linear opioid pentapeptide enkephalin analogues, suggesting that the Phe3 tetrapeptide side chain and the Phe4 pentapeptide side chain interact with the same delta receptor binding subsite.

Topics: Oligopeptides; Opioid Peptides; Peptides, Cyclic; Receptors, Opioid; Receptors, Opioid, delta; Substrate Specificity

As many as 47 amphibian and mammalian, natural and non-natural opioid peptides have been examined in guinea-pig ileum (GPI) and mouse vas deferens (MVD) preparations. The great value of these extremely simple and accessible tissue models in the identification, isolation and purification of endogenous opioid peptides, in studying structure/activity relationships, and in determining selectivity of the peptide molecules for the various opioid receptors, especially delta- and mu-receptors, is emphasized.

Topics: Amino Acid Sequence; Animals; Biological Assay; Caseins; Endorphins; Enkephalins; Guinea Pigs; Ileum; Isometric Contraction; Male; Mice; Molecular Sequence Data; Muscle, Smooth; Oligopeptides; Opioid Peptides; Receptors, Opioid, delta; Receptors, Opioid, mu; Structure-Activity Relationship; Vas Deferens

The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal3 and 2-Nal3 substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal3 side chain but only the delta receptor can readily accept the more elongated 2-Nal3 side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3 substitutions, Cha3 substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [D-Pen2, D-Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.

Topics: Amino Acid Sequence; Animals; In Vitro Techniques; Kinetics; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Receptors, Opioid, delta; Receptors, Opioid, mu; Structure-Activity Relationship

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.

Topics: Amino Acid Sequence; Animals; Binding Sites; Brain; Cell Membrane; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

1. To elucidate the structural features required for selective and potent action of dermenkephalin at the delta-opioid receptor, a series of analogues of dermenkephalin and dermorphin were tested for their effectiveness in depressing electrically-evoked contractions of the vas deferens of the hamster (delta-opioid receptors) and the guinea-pig myenteric plexus-longitudinal muscle preparation (mu- and kappa-opioid receptors). 2. Dermenkephalin was more selective and more potent at delta-receptors than the delta-ligand [D-Pen2, D-Pen5]-enkephalin. The responses to dermenkephalin in the hamster vas deferens were increased by addition of peptidase inhibitors; the maximum effect was obtained with 3 microM thiorphan. 3. [L-Met2]-dermenkephalin had 0.2% and [L-Ala2]-dermorphin 0.01% of the agonist activity of the corresponding endogenous peptides which have D-amino acids in position 2. The pharmacological activity of these analogues was unaffected by inhibition of peptidases. This emphasizes the role that the D-configuration plays in determining the bioactive folding of these highly active peptides. 4. Dermenkephalin-(1-6)-NH2 was more potent at delta-receptors than at mu-receptors whereas, dermenkephalin-(1-4)-NH2 is a selective mu-agonist, having no activity at delta-receptors. 5. Substitution of the C-terminal tripeptide of dermorphin with the C-terminal tripeptide of dermenkephalin abolished the mu-receptor preference of dermorphin. The resulting hybrid peptide, Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2 was as potent as dermenkephalin at delta-receptors. A shift towards a preference for delta-receptors was obtained when the C-terminal tetrapeptide of dermorphin was replaced by the C-terminal tetrapeptide of dermenkephalin. 6. Substitution of Asp by Asn in position 7 of dermenkephalin caused an increase in mu-receptor potency and a decrease in delta-receptor potency, resulting in a 20 fold decrease in mu-receptor selectivity. Dermenkephalin-(1-6)-NH2 and [Asn7]-dermenkephalin have almost identical delta-receptor agonist potencies and ratios of IC50 in the myenteric plexus to IC50 in the hamster vas deferens. 7. The results obtained emphasise the importance of a negative charge at the C-terminus of dermenkephalin for selectivity at the delta-opioid receptor. Furthermore, the hydrophobic residues Leu5 and Met6 may be critical in ensuring tight binding to the receptor which results in high agonist potency.

Topics: Amino Acid Sequence; Amino Acids; Animals; Biological Assay; Cricetinae; Guinea Pigs; In Vitro Techniques; Male; Mesocricetus; Mice; Molecular Sequence Data; Muscle, Smooth; Myenteric Plexus; Oligopeptides; Opioid Peptides; Peptides; Protease Inhibitors; Protein Conformation; Receptors, Opioid; Vas Deferens

The effects of intracerebroventricular administration of dermorphin (mu agonist) and dermenkephalin (delta agonist) were studied at the electrocorticographic (ECoG) and electromyographic (EMG) levels in free-moving rats. A very low dose of dermorphin (125 pmol) induced ECoG spiking, occasional myoclonic jerks, wet-dog shakes and catalepsy. In addition, electrical seizures were triggered with doses of 250 to 500 pmol. These signs were reversed by an i.p. injection of naloxone (1 mg kg-1). However, no epileptic-like phenomena were seen after administration of dermenkephalin up to 1 nmol. These results indicate that mu but not delta receptors are involved in the epileptogenic effect of intraventricularly administered opiates.

Topics: Animals; Cerebral Cortex; Cerebral Ventricles; Electroencephalography; Electromyography; Epilepsy; In Vitro Techniques; Injections, Intraventricular; Oligopeptides; Opioid Peptides; Rats; Rats, Inbred Strains

The naturally occurring amphibian skin peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) are highly potent and selective agonists at the mu- and the delta-opioid receptors, respectively. For peptides synthesized by animal cells, they have a rather peculiar structural feature of containing a D-amino acid residue in their sequence which imparts biological activity on them. The cloned cDNA encoding the prodermorphin precursor contains the usual alanine and methionine codons at positions where D-alanine and D-methionine are present in the mature products. In this study, dermorphin precursor was characterized in extracts from amphibian skin by antisera recognizing distinct epitopes within the predicted structure of pro-dermorphin. Proteolytic digestion of purified endogenous pro-dermorphin generated a peptide containing a D-alanine in position 2, identified as prepro-dermorphin-(80-89), i.e. Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala. In addition, analysis of skin extracts by enzyme immunoassays coupled to high performance liquid chromatography separations revealed the presence of, besides dermenkephalin, novel dermenkephalin-related peptides, i.e. [L-Met2]dermenkephalin, dermenkephalin-OH, and [Met(O)6]dermenkephalin. [L-Met2]dermenkephalin was present in frog skin in a concentration of about 100 times that of dermenkephalin. These observations confirm that, despite the presence of D-amino acid residues, dermorphin and dermenkephalin are genuine products of post-translational processing of a ribosomally made precursor. They suggest that D-Ala and D-Met develop from a dehydrogenation/hydrogenation stereoinversion of their corresponding L isomers incorporated into pro-dermorphin, a process that occurs with low efficiency at an early stage of biosynthesis.

Topics: Alanine; Amino Acid Sequence; Amphibians; Animals; Carboxypeptidase B; Carboxypeptidases; Chromatography, High Pressure Liquid; Cross Reactions; Immunoenzyme Techniques; Mass Spectrometry; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Protein Precursors; Protein Processing, Post-Translational; Trypsin

Comprehensive energy calculations were applied to four opioid-related peptides with different receptor selectivities, namely the delta-selective dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, DRE), the mu-selective dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DRM) and their "hybrid" peptides DRM/DRE (Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2) and DRE/DRM (Tyr-D-Met-Phe-His-Tyr-Pro-Ser-NH2). It was shown that the N-terminal tripeptide "mu-messages" in the delta-selective ligands DRE and DRM/DRE can possess similar low energy space arrangements of their functionally important elements (the N-terminal alpha-amino group and the aromatic moieties of Tyr and Phe), but that these are different from the space arrangement of these moieties in mu-selective DRM and DRE/DRM. These results suggest that the C-terminal tripeptide "delta-address" in DRE may influence the conformation of the "mu-message" in DRM. A refined model for the delta-receptor-bound conformation of DRE is proposed based on these calculations which is similar to that previously suggested for the cyclic delta-selective peptide [D-Pen2, D-Pen5]enkephalin (DPDPE). This model also has partial correspondence with the structure of the delta-selective alkaloid naltrindole.

Topics: Amino Acid Sequence; Analgesics, Opioid; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Protein Conformation; Receptors, Opioid; Receptors, Opioid, mu; Stereoisomerism; Structure-Activity Relationship

Dermorphin (Tyr-D.Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and dermenkephalin (Tyr-D.Met-Phe-His-Leu-Met-Asp-NH2), two powerful opioid peptides issued from a common biosynthetic precursor, were recently isolated from the skin of the frog phyllomedusa sauvagii. Since many amphibian's skin secretory peptides or their homologues are found in the mammalian central nervous system and gastrointestinal tract, dermorphin and dermenkephalin may have counterparts in mammals. We have prepared antibodies directed against dermorphin, dermenkephalin and the spacer sequence lying between them in the progenitor form and developed sensitive enzyme immunoassays that detect under the picogram level to verify the occurrence of these peptides in rat tissues. Immunocytochemical studies of rat brain sections revealed a similar distribution of immunoreactivities both at the regional and the cellular levels when either one of these three anti-sera was used. Characterization of the immunoreactive peptides was achieved with molecular sieve filtration followed with Reverse Phase High Performance Liquid Chromatography of various rat tissues extracts. Identification was achieved by immunological analysis and chromatographic comparison with synthetic peptides. Immunoreactive materials corresponding to dermorphin, dermenkephalin and the spacer peptide were detected in either brain, stomach or intestine, indicating processing of the dermorphin precursor in these tissues. Immunoreactive species of higher Mr were also detected in all three tissues and may represent extended forms or homologous peptides.

Topics: Amino Acid Sequence; Animals; Brain; Cross Reactions; Endorphins; Gastric Mucosa; Immunoenzyme Techniques; Immunohistochemistry; Intestinal Mucosa; Male; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Protein Precursors; Rats; Rats, Inbred Strains; Tissue Distribution

Deltorphin is an opioid peptide with the sequence H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the delta-opioid receptor and the similarity of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H-NMR study in two different solvent systems showed that the conformational preferences of the N-terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.

Topics: Amino Acid Sequence; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Protein Conformation; Receptors, Opioid; Receptors, Opioid, delta

Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 is an extraordinarily potent and highly mu-selective opioid heptapeptide isolated from amphibian skin. It is unique among peptides synthesized by animal cells in having an amino acid residue in the D-configuration. At least two different preprodermorphin cDNAs were cloned from skin of Phylomedusa sauvagei; their predicted amino acid sequences contained four to five homologous repeats of 35 amino acids, each repeat including one copy of the dermorphin progenitor sequence. Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly, flanked by Lys-Arg at the amino end and by Glu-Ala-Lys-Lys at the carboxyl end [Science (Wash. D. C.) 238:200-202 (1987)]. The D-Ala in position 2 in dermorphin is encoded by a usual Ala codon in the precursor sequence. Of the two prodermorphin molecules, one has a dermorphin copy replaced with a distinct heptapeptide same processing signals. Assuming the same pathway as for the release of dermorphin, processing of this precursor may yield, beside dermorphin, a copy of a new peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2. We have synthetized this peptide together with its (L-Met2)-counterpart and evaluated their respective opioid receptor selectivity in the mouse vas deferens and guinea pig ileum assays and in rat brain membrane binding assays. Overall, the data collected demonstrate that the putative prodermorphin product Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 named dermenkephalin, behaves as a potent delta opioid agonist exhibiting high affinity and high selectivity for the delta opioid receptor. Prodermorphin, thus, offers a surprising example of an opioid biosynthetic precursor that might simultaneously generate highly potent and fully selective agonists for the mu- (morphine) and the delta (enkephalin) opioid receptors, respectively. In addition, because dermenkephalin has no structural features in common with the sequence of all the hitherto known opioid peptides, it should be a useful tool for identifying conformational determinants for high affinity and selective binding of opioids to the delta receptor.

Topics: Amino Acid Sequence; Analgesia; Animals; Binding, Competitive; Brain; Cell Membrane; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Kinetics; Male; Mice; Oligopeptides; Opioid Peptides; Pain; Rats; Receptors, Opioid; Receptors, Opioid, delta

Dermorphin and dermenkephalin are D-aminoacid containing peptides generated from processing of the plurifonctional biosynthetic precursor pro-dermorphin. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM) and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), are among the most selective and potent agonists described respectively for the mu- and delta-opioid receptors. In order to identify determinants of selectivity and high-affinity receptor binding of dermorphin and dermenkephalin, a series of analogs was investigated for their affinity at the mu- and delta-receptors in the brain. The tetrapeptide amino end of both DRM and DREK were found to display high affinity and selectivity towards mu-receptors. Substitution of the C-terminal tripeptide of DREK with that of DRM reversed the receptor selectivity of DREK from delta to mu. Replacement of the C-terminal tripeptide of DRM with the C-terminal counterpart of DREK shifted the selectivity of DRM from mu to delta. These data emphasize the critical contribution of the carboxy end of DREK to delta-selectivity. They further suggest that the potent mu-address lying in the N terminus of DREK is overwhelmed by the powerful delta-directing ability of the carboxy end. Unlike DREK, the C-terminus of DRM is not involved in opioid receptor sites selection but is important insofar as it serves to stabilize interactions of DRM with the mu-receptor binding site.

Topics: Animals; Binding, Competitive; Brain; Oligopeptides; Opioid Peptides; Protein Conformation; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

Processing of the polyprotein precursor pro-dermorphin generates two distantly related D-amino acid-containing peptides, dermorphin and dermenkephalin, which are among the most selective high affinity agonists described, respectively, for the mu- and delta-opioid receptors. Dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, is a linear, potentially flexible peptide devoid of structural homology with either enkephalins, endorphins, or dynorphins and, as such, represents a useful tool for identifying determinants of high affinity and selective binding of opioids to the delta-receptor. A series of selected dermenkephalin analogs and homologs was investigated for affinity at the mu- and delta-sites in the brain. Whereas dermenkephalin has high affinity and specificity for the delta-opioid receptors, its tetrapeptide amino end, dermenkephalin-[1-4]-NH2 binds almost exclusively at the mu-receptors. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, is only marginally more selective for the u-sites than is dermenkephalin-[1-4]-NH2. Using dermorphin-dermenkephalin peptide hybrids and C-terminal deletion analogs of dermenkephalin, we showed the critical role that the C-terminal residues Met6 and Asp7 play in specifying correct addressing of dermenkephalin toward delta-receptors. The potent mu-deteminant located within the amino end of dermenkephalin is over-whelmed by the powerful delta-directing ability of the carboxy end. The negatively charged side chain of Asp7 makes a significant contribution to the delta-addressing ability of the C-terminal region, a finding consistent with Schwyzer's membrane selection model (Schwyzer, R. (1986) Biochemistry 25, 6335-6342). The Leu residue in position 5 and D-configuration about the alpha-carbon of Met2 were found to be of crucial importance for high affinity binding to delta-receptors. Whereas the Met residue in position 6 in dermenkephalin could safely be oxidized or replaced with D-Met, oxidation of Met2 led to deleterious effects, this analog being 1/100 as potent as dermenkephalin at delta-sites. Overall, the data collected demonstrate that highest levels of selectivity and affinity for the delta-opioid receptors can be achieved with small-sized, potentially flexible, linear peptides and further support the model according to which, in addition to optimum accommodation at the receptor, selection for delta-receptors is reduced by the effective positive charge of the molecule. Dermenkephalin may provide a starting point for

Topics: Amino Acid Sequence; Animals; Male; Molecular Sequence Data; Oligopeptides; Opioid Peptides; Peptide Fragments; Protein Conformation; Protein Multimerization; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sequence Homology, Nucleic Acid; Structure-Activity Relationship