Opioid infidelity: novel opioid peptides with dual high affinity for delta- and mu-receptors.

Deltorphins represent the paragon of delta-opioid-receptor ligands of natural origin, since they exceed the affinity and selectivity of the endogenous enkephalins by orders of magnitude. A series of opioid peptides have been developed in which the change in a single amino acid causes an extraordinary increase in mu-receptor binding while maintaining high affinity for the delta-receptor. The peptides appear to have a similar extended conformation in solution with a type-I beta-turn in the N-terminus region, suggesting that tertiary architecture plays a pivotal role in enabling the peptide to bind indiscriminately to mu- and delta-receptors. These dual-affinity peptide ligands can serve to mask delta- and mu-receptors while mapping kappa-receptors in the nervous system, to provide an understanding of the differences and similarities in the structure of the binding domains of delta- and mu-receptors, and might lead to a comprehensive new regime for the clinical management of acute and chronic pain.

What peptides these deltorphins be.

The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.

Phe3-substituted analogues of deltorphin C. Spatial conformation and topography of the aromatic ring in peptide recognition by delta opioid receptors.

In order to study the contribution of the electronic, hydrophobic, and conformational properties of the amino acid residue at position 3 in deltorphin C on binding to delta and mu opioid receptors, a series of 5- and 6-membered ring and bicyclic amino acid replacements at position 3 were prepared by solution synthesis methods. In general, the substitutions were deleterious for high delta affinity (Ki delta) and delta selectivity (Ki mu/Ki delta). However, several notable exceptions were recognized: peptides containing the constrained, bicyclic structures Aic3 and (R or S) Atc3 enhanced delta affinity, but only the latter increased delta selectivity 4-fold (= 2475) relative to deltorphin C (= 661); at the other extreme, delta affinity of N alpha MePh3 fell 900-fold. Bioassays of [N alpha MePhe3]-, [(R or S)C alpha MePhe3]-, [Tic3]-, [Aic3]-, and [(R or S) Atc3]deltorphin C using guinea pig ileum (GPI) and mouse vas deferens (MVD) for mu and delta bioactivity, respectively, revealed a significant correlation (r = 0.916) between MVD bioactivity and delta binding in brain membranes. [(R or S)Atc3]deltorphin C also exhibited the highest biological selectivity (GPI/MVD) (= 3,522), which was 3-fold greater than that observed for deltorphin C. Molecular modelling of [N alpha MePhe3]- and [(S)Atc3]deltorphin C established that these amino acid replacements for Phe3 produce alterations in the backbone (phi,psi) and side-chain (chi 1,chi 2) dihedrals which critically affect the flexibility of the peptide and possibly limit accessible conformations for its alignment within the delta opioid receptor. The data provide evidence that the delta receptor is sensitive to changes in the composition, conformation, and orientation of the side chain of residue 3 of a linear opioid heptapeptide.

Design and synthesis of 1-aminocycloalkane-1-carboxylic acid-substituted deltorphin analogues: unique delta and mu opioid activity in modified peptides.

Deltorphin analogues were substituted by a series of achiral C alpha,alpha-dialkyl cyclic alpha-amino acids (1-aminocycloalkane-1-carboxylic acids, Ac chi c, where chi = a hexane, pentane, or propane cycloalkane ring) in position 2, 3, 4, or 2 and 3 in deltorphin C, and in position 2 in [Ac6c2,-des-Phe3]deltorphin C hexapeptide. Receptor assays indicated that even though Ac6c2 and Ac6c3 exhibited a diminished Ki delta by ca. 20-fold (2.5-3.3 nM) relative to deltorphin C (Ki delta = 0.15 nM), selectivity was marginally elevated (Ki mu/Ki delta = 1250) or enhanced by about 70%, and both peptides fitted stringent iterative calculations for a two-site binding model (eta = 0.625 and 0.766, respectively, P < 0.0001). The disubstituted [Ac6c2,3]- or [Ac6c2,des-Phe3]deltorphin analogues yielded peptides with decreased Ki delta, such that the latter peptide was essentially inactive. The presence of Ac5c or Ac3c in place of Phe3 further diminished Ki delta (15.4 to 19.0 nM), yet delta selectivity only fell about one-half (Ki mu/Ki delta = 440 and 535, respectively), and only the former peptide fitted a two-site binding model (eta = 0.799). The replacement of Asp4 by Ac6c, Ac5c, or Ac3c produced essentially nonselective analogues through the acquisition of high mu affinities (2.5, 0.58 and 0.27 nM, respectively) while maintaining high delta affinities (Ki delta = 0.045-0.054 nM) which were about 3-fold greater than that of deltorphin C. Using pharmacological assays in vitro (mouse vas deferens and guinea pig ileum), position 3-substituted analogues all indicated substantial losses in bioactivity, whereas substitution by 1-aminocycloalkanes at the fourth position retained high delta activity. In fact, the bioactivity of [Ac3c4]deltorphin C indicated a peptide with relatively weak delta selectivity, which was comparable to the observations with the receptor binding data. In summary, the data confirmed that (i) delta selectivity occurs in the absence of D-chirality at position 2, (ii) the aromaticity of Phe3 is replaceable by an achiral residue with a hydrophobic ring-saturated side chain, and (iii) the acquisition of dual high-affinity analogues occurs through the elimination of the anionic function at position 4 and replacement by an amino acid with a hydrophobic side chain.

Helix-inducing alpha-aminoisobutyric acid in opioid mimetic deltorphin C analogues.

The achiral symmetric alpha-aminoisobutyric acid (Aib) replaced the critical N-terminal residues of the amphibian skin opioid deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) without detriment to the physicochemical requirements for delta opioid receptor recognition. Substitutions by the alpha, alpha-dialkyl amino acid in place of D-Ala2 or Phe3, or both, exhibited high delta receptor affinity (Ki delta = 0.12-3.6 nM) and 5-9-fold greater selectivity (Ki mu/Ki delta = 5000-8500) than the parent compound. This is the first definitive demonstration that the D-chirality of alanine and the aromaticity of phenylalanine are replaceable by an achiral alpha, alpha-dialkylated residue without detrimental effects on ligand binding. Incorporation of the mono-alpha-alkyl amino acid L- or D-Ala at the third position also produced highly selective delta ligands (Ki mu/Ki delta = 2000-3500), albeit with reduced delta affinities (Ki delta = 6-15 nM). Replacement of the anionic residue Asp4 by Aib yielded an opioid peptide that fit two-site binding models for the delta receptor (eta = 0.763; P < 0.0001) and displayed dual high affinity for both delta and mu receptors, emphasizing the repulsive effect by a negative charge at mu receptor sites and the insignificance of Asp for delta affinity. Molecular dynamics conformation analyses suggested that Aib residues caused distinct changes in deltorphin C secondary structure when substituted for D-Ala2, Asp4, and simultaneously D-Ala2 and Phe3 but not when substituted for Phe3. These conformational changes might be critical factors for the proper orientation of reactive constituents of residues in the N-terminal region of deltorphin C. Disparities between binding data and functional bioassays of [Aib3] indicated that Phe3 was required for bioactivity in mouse vas deferens but not for interaction with delta opioid receptors in rat brain membranes.

Evolution of the Dmt-Tic pharmacophore: N-terminal methylated derivatives with extraordinary delta opioid antagonist activity.

The delta opioid antagonist H-Dmt-Tic-OH (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) exhibits extraordinary delta receptor binding characteristics [Ki delta = 0.022 nM; Ki mu/Ki delta = 150,000] and delta antagonism (pA2 = 8.2; Ke = 5.7 nM). A change in chirality of Dmt at C alpha (1, 2, 6, 8, 10, 13) curtailed delta receptor parameters, while replacement of its alpha-amino function by a methyl group (3) led to inactivity; Tyr-Tic analogues 4 and 11 weakly interacted with delta receptors. N-Alkylation of H-Dmt-Tic-OH and H-Dmt-Tic-Ala-OH with methyl groups produced potent delta-opioid ligands with high delta receptor binding capabilities and enhanced delta antagonism: (i) N-Me-Dmt-Tic-OH 5 had high delta opioid binding (Ki delta = 0.2 nM), elevated delta antagonism on mouse vas deferens (MVD) (pA2 = 8.5; Ke = 2.8 nM), and nondetectable mu activity with guinea pig ileum (GPI). (ii) N,N-Me2-Dmt-Tic-OH (12) was equally efficacious in delta receptor binding (Ki delta = 0.12 nM; Ki mu/Ki delta = 20000), but delta antagonism rose considerably (pA2 = 9.4; Ke = 0.28 nM) with weak mu antagonism (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:5640). N-Me-(9) and N,N-Me2-Dmt-Tic-Ala-OH (15) also augmented delta opioid receptor binding, such that 15 demonstrated high affinity (Ki delta = 0.0755 nM) and selectivity (Ki mu/Ki delta = 20132) with exceptional antagonist activity on MVD (pA2 = 9.6; Ke = 0.22 nM) and weak antagonism on GPI (pA2 = 5.8; Ke = 1.58 microM; GPI/MVD = 1:7180). Although the amidated dimethylated dipeptide analogue 14 had high Ki delta (0.31 nM) and excellent antagonist activity (pA2 = 9.9; Ke = 0.12 nM), the increased activity toward mu receptors in the absence of a free acid function at the C-terminus revealed modest delta selectivity (Ki mu/Ki delta = 1655) and somewhat comparable bioactivity (GPI/MVD = 4500). Thus, the data demonstrate that N,N-(Me)2-Dmt-Tic-OH (12) and N,N-Me2-Dmt-Tic-Ala-OH (15) retained high delta receptor affinities and delta selectivities and acquired enhanced potency in pharmacological bioassays on MVD greater than that of other peptide or non-peptide delta antagonists.

Further studies on the Dmt-Tic pharmacophore: hydrophobic substituents at the C-terminus endow delta antagonists to manifest mu agonism or mu antagonism.

Twenty N- and/or C-modified Dmt-Tic analogues yielded similar K(i) values with either [(3)H]DPDPE (delta(1) agonist) or [(3)H]N, N(Me)(2)-Dmt-Tic-OH (delta antagonist). N-Methylation enhanced delta antagonism while N-piperidine-1-yl, N-pyrrolidine-1-yl, and N-pyrrole-1-yl were detrimental. Dmt-Tic-X (X = -NHNH(2), -NHCH(3), -NH-1-adamantyl, -NH-tBu, -NH-5-tetrazolyl) had high delta affinities (K(i) = 0.16 to 1 nM) with variable mu affinities to yield nonselective or weakly mu-selective analogues. N, N-(Me)(2)Dmt-Tic-NH-1-adamantane exhibited dual delta and mu receptor affinities (K(i)delta = 0.16 nM and K(i)mu = 1.12 nM) and potent delta antagonism (pA(2) = 9.06) with mu agonism (IC(50) = 16 nM). H-Dmt-betaHTic-OH (methylene bridge between C(alpha) of Tic and carboxylate function) yielded a biostable peptide with high delta affinity (K(i) = 0.85 nM) and delta antagonism (pA(2) = 8.85) without mu bioactivity. Dmt-Tic-Ala-X (X = -NHCH(3), -OCH(3), -NH-1-adamantyl, -NHtBu) exhibited high delta affinities (K(i) = 0.06 to 0.2 nM) and elevated mu affinities (K(i) = 2.5 to 11 nM), but only H-Dmt-Tic-Ala-NH-1-adamantane and H-Dmt-Tic-Ala-NHtBu yielded delta receptor antagonism (pA(2) = 9.29 and 9.16, respectively). Thus, Dmt-Tic with hydrophobic C-terminal substituents enhanced mu affinity to provide delta antagonists with dual receptor affinities and bifunctional activity.

Frog skin opioid peptides: a case for environmental mimicry.

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.

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist.

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective delta opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3',5'-I2)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the delta opioid receptor whereas its binding at mu and kappa opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue ¿35SGTPgammaS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study delta opioid receptors in vitro and in vivo.

Opioid pseudopeptides containing heteroaromatic or heteroaliphatic nuclei.

In lieu of H-Dmt-Tic-OH, H-Dmt-analogues included 2-amino-3(1H-benzoimidazol-2-yl)-propionic acid, N(Bzl)Gly, L-octahydroindole-2-carboxylic acid, [3S-(3alpha,4abeta, 8abeta)]-decahydro-3-isoquinoline carboxylic acid, benzimidazole-, pyridoindole- or spiroinden-derivatives, or C-terminally modified. L- or D-Ala, Sar, or Pro were spacers between aromatic nuclei. Only H-Dmt-(Xaa-)-pyridoindole exhibited high affinities with delta and mu antagonism. The peptides competed equally against [3H]DPDPE (delta agonist) or [3H]N,N(CH3)2-Dmt-Tic-OH (delta antagonist) signaling a single delta binding site. The data confirm the importance of Tic for delta affinity and antagonism, while heterocyclic or heteroaliphatic nuclei, or spacer exert effects on mu- and delta-receptor properties.

Inhibition of human multidrug resistance P-glycoprotein 1 by analogues of a potent delta-opioid antagonist.

Analogues Dmt-Tic (2',6'-dimethyl-L-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) pharmacophore, a potent delta-opioid receptor antagonist, inhibited hMDR1 P-GP expressed in a G-185 fibroblast cell line in a manner similar to verapamil. N,N(Me)2-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-Ala-NH-1-adamantane and N,N(Me)2-Dmt-Tic-NH-tBut were highly effective inhibitors. Weaker inhibition was observed with N,N(Et)2-Dmt-Tic-OH, H-Dmt-Tic-Ala-NH-tert-butyl amide and cyclo(Dmt-Tic). Results demonstrate that N- and C-terminal hydrophobic/lipophilic analogues of the Dmt-Tic pharmacophore inhibit hMDR1 and point to a potential role as chemosensitizing agents in chemotherapy for cancers containing hMDR1.

Relationship between receptor affinity and topography of N-terminally extended and bridged [Tyr1-->Asp4]deltorphin C analogues: novel probes for the delta-opioid receptor.

Receptor binding of N-terminally extended Tyr1 deltorphin C analogues diminished delta and mu affinities, but with only a moderate loss in delta selectivity. Pseudopeptide bridged [Tyr1-->Aps4]deltorphin C analogues drastically decreased delta affinity to yield peptides with poor delta selectivity. Low energy conformers of the peptides revealed that the bridge modifies the spatial orientation of the backbone of the N- and C-terminal sequences with respect to deltorphin C. The data indicate that the delta receptor site can accommodate an opioid peptide containing an N-terminal aliphatic extension on amino-Tyr1, but not a heptapeptide conformationally constrained between residues 1 and 4.

Opioid receptor selectivity alteration by single residue replacement: synthesis and activity profile of [Dmt1]deltorphin B.

The single amino acid replacement of 2',6'-dimethyl-L-tyrosine in deltorphin B (H-Dmt-D-Ala-Phe-Glu-Val-Val-Gly-NH2) yielded high affinity for mu- and delta-binding sites. [Dmt1]Deltorphin B lacks activity at kappa-opioid binding sites. Bioactivity in vitro with guinea-pig ileum confirmed that [Dmt1]deltorphin B interacted with mu-opioid receptors by reducing electrically induced contractions in a naloxone-reversible manner and was 150-fold more potent than morphine and comparable to [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO). The inhibition of spontaneous contractions of rabbit jejunum provided evidence for delta-opioid receptor interaction. Analgesia (hot plate and tail flick tests) revealed that [Dmt1]deltorphin B was 180- to 200-fold more potent than morphine. Pretreatment with naloxone, naltrindole or H-Dmt-Tic-Ala-OH (a highly selective delta-opioid receptor antagonist) prevented [Dmt1]deltorphin B antinociception. Thus, [Dmt1]deltorphin B exhibited remarkably high dual affinity and bioactivity toward delta- and mu-opioid receptors.

Inverse agonism by Dmt-Tic analogues and HS 378, a naltrindole analogue.

The potent delta-opioid receptor antagonist H-2',6-L-tyrosine(Dmt)-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic-OH) exhibited partial inverse agonism (EC(50)=6.35 nM, E(max)=-18.87%) for [35S]GTPgammaS binding and H-Dmt-Tic-NH(2) was a neutral antagonist (no effect up to 30 microM). In contrast N,N(CH(3))(2)-Dmt-Tic-NH(2) was a full inverse agonist (EC(50)=2.66 nM, E(max)=-35.95%) similar to ICI 174864 ([N,N-diallyl-Tyr(1),Aib(2,3),Leu(5)]enkephaline) but with a 3.5-fold higher EC(50). In comparison, naltrindole was a neutral antagonist while its analogue HS 378 was a partial inverse agonist (E(max)=-12.99%).

Dmt-Tic-OH a highly selective and potent delta-opioid dipeptide receptor antagonist after systemic administration in the mouse.

Dmt-Tic-OH (DTOH) and Dmt-Tic-Ala-OH (DTAOH), effective antagonists in vitro, represent a new potent opioid dipeptides for the delta-opioid receptor (Ki delta of 0.022 nM and a selectivity, Ki mu/Ki delta, of 150,000 for DTOH; Ki delta of 0.285 nM and a selectivity Ki mu/Ki delta, of 20,4 for DTAOH). In the present study we considered the pharmacological activity of these two new delta opioid peptide receptor antagonists in vivo. Therefore, we have evaluated their possible antagonistic activity against the antinociception induced by the highly selective delta opioid receptor agonist, [D-Ala2]deltorphin II (DEL). Furthermore, these two delta opioid peptide receptor antagonists were injected centrally or peripherally in order to assess their ability to act also after systemic administration. Concurrent i.c.v. injection of DTOH or DTAOH (0.5-1.0-2.0 nM) with DEL (5 nmol) induced a significant reduction of DEL antinociception. By contrast, while DTOH (10-20-40 mg/kg) administered peripherally (i.p., s.c. or i.v.) was also able to reduce DEL antinociception, DTAOH failed. The present results indicate that DTOH is the first opioid dipeptide with delta antagonist activity after systemic administration and it could be important in the clinical and therapeutic applications.

Lead-contaminated drinking waters in the public schools of Philadelphia.

UNLABELLED: Lead exposure is a preventable environmental health concern. Young children between the ages of 1 to 6 are most susceptible to its clinical effects. This article reports the results of lead level determinations in the drinking water of Philadelphia's public school buildings and remediation efforts aimed at dealing with this public health concern. METHODS: Water samples were collected from drinking sources in 292 school buildings in Philadelphia from May 2000 through January 2001. These samples were collected and sent to reference laboratories for determination of lead levels. RESULTS: A total of 42.5% (124) of schools had water lead levels not exceeding the action level of 20 ppb, of which 3.1% had nondetectable levels or levels less than 5 ppb. A total of 28.7% of buildings had water lead levels ranging from 20 to 50 ppb, 11.6% had levels between 50-100 ppb, and 17.1% had water lead levels of 100 ppb or more. CONCLUSION: A total of 57.4% of Philadelphia's public school buildings had water lead levels exceeding the Environmental Protection Agencies (EPA) action level of 20 ppb, and 28.7% of school buildings had water with mean lead levels in excess of 50 ppb. Depending on the volume of water consumed, drinking water from school buildings may be a significant source of lead exposure for children in their formative years of development. Although Philadelphia's public school buildings were evaluated, lead-contaminated drinking water in schools is not only an urban concern. School buildings in suburban and rural areas may have similar water lead levels, and testing programs are desirable.

Indole-3-acetic Acid Oxidase from Peas: I. Occurrence and Distribution of Peroxidative and Nonperoxidative Forms.

Tissues of etiolated pea seedlings variety Alaska were examined for the presence of peroxidative and nonperoxidative forms of indoleacetic acid (IAA) oxidase. Enzymes were extracted in a sequence involving acetone powder preparation from pea tissues, buffer extraction of the powder, ammonium sulfate precipitation, dialysis, lyophilization, and acrylamide gel electrophoresis. Electrophoretically separable proteins were assayed for IAA oxidase activity with the Salkowski test, and peroxidase activity was based on the color reaction with benzidine and H(2)O(2). Each tissue examined contained several nonperoxidative IAA oxidases. No tissue contained more than three peroxidative IAA oxidases, whereas the plumule hooks (a tissue with a high IAA oxidase activity) contained no detectable peroxidases. The results indicate that nonperoxidative IAA oxidases might play a major role in the regulation of IAA content in pea seedlings.

Molecular dynamics conformations of deltorphin analogues advocate delta opioid binding site models.

Multi-site binding models for the delta opioid receptor were studied in vitro with [3H]DPDPE as the labeled ligand using analogues of deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) altered at position 4. Modifications included a change in chirality (L- to D-Asp4), increased length of the anionic side-chain (Glu4), elimination of the charged group (Abu4), addition of an anionic group (Gla4), and change in backbone conformation (Pro4). All of the peptides had relatively high delta affinities (0.09 to 1.15 nM); the major variability in delta selectivity resided in changes in mu affinities (1.6 to 530 nM). Three analogues (Glu4, D-Asp4 and Pro4) revealed better fits to two-site binding models (Hill coefficients < 0.850 with narrow 95% confidence intervals and P < 0.0001). Deltorphin C and analogues containing Gla4 and Abu4 (which were weakly delta selective), as well as deltorphin A (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2), fitted one-site binding models. Molecular dynamics simulations performed on deltorphin C and Abu4 exhibited similarities in the tertiary structure of their low energy conformers, while differing from the three-dimensional structures of the analogues containing Glu4, D-Asp4 and Pro4 substitutions. The data provide support that the three-dimensional architecture of an opioid peptide is an important factor in the designation of delta opioid receptor subtypes.

Prerequisite for His(4) in deltorphin A for highδ opioid receptor selectivity.

Analysis of deltorphin A position 4 analogues included: backbone constrained N (α) MeHis, spinacine (Spi), N (α) MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofL- andD-His(4) enantiomers. Highδ selectivity was lost with the following replacements: N (α) MeHis(4), N (α) MePhe(4) and Phg(4) reducedδ binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi(4) or Tic(4) increasedµ affinity. Imidazole methylation of His(4) marginally affected opioid binding and doubledδ selectivity; alkylatedD-His(4)-derivatives generally maintainedδ selectivity in spite of decreasedδ affinities. Thus, His(4) imidazole preservesδ selectivity by facilitating highδ binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His(4) imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr(1) suggestive that this conformation might contribute to highδ affinity and selectivity.