Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the μ- and §-types. They included [D-Cys², L-Cys⁵]- and [D-Cys², D-Cys⁵]-enkephalinamides showing moderate preference for preceptors, the selective compounds [D-Pen², L-pen⁵] and [D-Pen², D-Pen⁵]-enkephalins and Tyr-D-Lys-Gly-Phe analogue possessing very high affinity to receptors of the μ-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors

Models of μ- and δ-receptor-bound backbone conformations of enkephalin cyclic analogues containing Phe⁴ were determined by comparing geometrical similarity among the previously found low-energy, backbone structures of -enkephalinamide, -enkephalinamide, -enkephalin and -enkephalin. The present μ-receptor-bound conformation resembles a β-I bend in the peptide backbone centred on the Gly³-Phe⁴ region. Two slightly different models were found for the δ-receptor-bound conformation; both of them are more extended than the μ-receptor-bound conformation and include a γ-turn (or a γ-like turn) on the Gly³ residue. Energetically favourable rotamers of Tyr and Phc side chains were also determined for the μ- and δ-conformations. The present models of μ- and δ-conformations share geometrical similarity with the low-energy structures of Leu-enkephalin and the analogue.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

Solution conformations of the peptide backbone for DPDPE and its β-MePhe4-substituted analogs

The solution structures of DPDPE, a conformationally restricted pentapeptide with the sequence H-Tyr¹-d -Pen²-Gly³-Phe⁴-d -Pen⁵-OH, and its four β-MePhe⁴-substituted analogs were examined by a combined approach including the NMR measurements in DMSO and water as well as independent energy calculations. It was concluded that several low energy conformers of DPDPE backbone satisfy the NMR data obtained in this study as well as in previous studies by other authors. These possible solution conformers of DPDPE in both DMSO and water share virtually the same type of cyclic backbone structure, with the Gly³ residue in a conformation close to a γ-turn, and the Phe⁴ residue in a conformation close to α-helical torsion angles. They differ in the space arrangements of the flexible Tyr¹ moiety. The solution structures of the β-MePhe⁴-substituted analogs of DPDPE are interesting. For analogs with an S-configuration at the C^α atom in the Phe⁴ residue, the cyclic backbone conformations resemble those of DPDPE itself, whereas for analogs with an R-configuration at the C^α atom, the backbone conformation is somewhat different. This observation is in line with the high biological potencies and selectivities displayed by the former compounds but not by the latter ones. It was noted also that as far as the peptide backbone conformers are concerned, some of the possible DPDPE conformers in water are similar to the previously suggested model for the δ-receptor-bound conformation of DPDPE, becoming virtually identical to this conformation by rotating the side chains of the Tyr¹ and the Phe⁴ residues.     

A chimeric opioid peptide with mixed μ agonist/δ antagonist properties

Abstract: There is evidence to indicate that opioid compounds with mixed μ agonist/δ antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective μ agonist H‐Dmt‐D‐Arg‐Phe‐Lys‐NH₂ ([Dmt¹]DALDA) (Dmt = 2′,6′‐dimethyltyrosine) and the potent and selective δ antagonist H‐Tyr‐TicΨ[CH₂‐NH]Cha‐Phe‐OH (TICP[Ψ]) (Cha = cyclohexylalanine), connected ‘tail‐to‐tail’ via a short linker, was synthesized using a combination of solid‐phase and solution techniques. The resulting peptide, H‐Dmt→D‐Arg→Phe→Lys‐NH‐CH₂‐CH₂‐NH‐Phe←Cha[NH‐CH₂]ΨTic←Tyr‐H, showed the expected μ agonist/δ antagonist profile in the guinea‐pig ileum and mouse vas deferens assays. Its μ and δ receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Binding to opioid receptors of enkephalin derivatives taking α-helical conformation and its dimer

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the σ-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the μ-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-GIy-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)₂-OH (2) showed 51-and 96-fold decreases in affinities for δ- and μ-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to α-helical conformation, which might not be compatible with topological requirements of δ- and μ-receptors. A dimer, Tyr-Gly-GIy-Leu-Phe-(Lys-Aib-Leu-Aib)₂-Lys(X)-Aib-OCH₃ (X = Tyr-Gly-Gly-Phe-Leu-, (4)), showed 2.5- and 3.0-fold increases in affinities respectively for δ- and μ-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between δ- and μ-receptors in bovine brain membranes.     

Effects of intracerebroventricular injection of opioid peptides selective for μ, δ and κ receptors on discriminative stimulus properties of pentazocine in the rat

The present study was designed to investigate the effects of centrally administered morphine and opioid peptides on the discriminative stimulus properties of pentazocine in the rat. Rats were trained to discriminate 3 mg/kg (s.c.) of pentazocine from vehicle in a shock avoidance paradigm. A 3 mg/kg (s.c.) dose of pentazocine produced stimulus effects in common with those induced by a training dose (3 mg/kg, s.c.) of pentazocine. Morphine (0·1–3 μg, i.c.v.) produced a dose-dependent increase in responding appropriate for pentazocine lever. The μ-selective opioid receptor agonist [D-Ala²,NMePhe⁴,Gly-ol] enkephalin (DAMGO) (0·0003–0·03 μg, i.c.v.) generalized to pentazocine cue. d -Pen², l -Pen⁵] enkephalin (DPLPE) (3 and 10 μg, i.c.v.), a δ-selective opioid receptor agonist, produced partial generalization to pentazocine cue. However, the κ-selective opioid receptor agonist dynorphin A-(1–13) (3 and 10 μg, i.c.v.) did not generalize to pentazocine cue. The pentazocine-like discriminative stimulus effects of morphine (3 μg, i.c.v.) and DAMGO (0·03 μg, i.c.v.) were fully reversed by intracerebroventricular injection of the μ-selective opioid receptor antagonist β-funaltrexamine (5 μg, i.c.v.). These results suggest that μ-opioid receptors play a major role in the discriminative stimulus effects of pentazocine, while δ-opioid receptors only partially contribute to them. © 1998 John Wiley & Sons, Ltd.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.     

Folded conformations of the δ-selective opioid dermenkephalin with head-to-tail interactions. A simulated annealing study through NMR restraints

Despite similar tripeptide N-termini, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ- and δ-opioid receptors, respectively. Structure-activity relationship studies have shown that the N-terminal tripeptide, Tyr-D-Xaa-Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ- and δ-receptors while the nature and/or the conformation of the C-terminus His-Leu-Met-Asp-NH₂ of dermenkephalin are responsible for addressing the peptide to the δ-receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ-receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two-dimensional ¹H-NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty-four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues, S-shaped structures, mean planes of N-terminal and C-terminal moieties almost at right angles to each other, a C-terminus region above the plane of the N-terminal region and g^? as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (t or g^?) and proximity between Tyr and Asp, or Tyr and the C-terminus. In contrast to previous models, practically no β-turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ-turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ-opioid receptor selectivity is discussed. © Munksgaard 1996.     

X-Ray structure of Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP-NH2), a highly potent μ-receptor selective opioid agonist. Comparisons with proposed model structures

Tyr-D-Tic-Phe-Phe-NH₂ (D-TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the μ-receptor. Its conformational features have been studied using a combination of solid-state (X-ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X-ray study showed two distinct conformers for D-TIPP, with the main differences lying in the orientation of the Tyr side-chain and the presence of both D-Tic(+) and D-Tic(—) conformations for the D-Tic residue. The peptide backbone is folded and stablized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D-Tic residue are found in the lowest-energy structures. Comparison of the X-ray and modeling results shows many similarities especially around the D-Tic residue. © Munksgaard 1997.     

Cyclic enkephalin analogs that are hybrids of DPDPE-related peptides and Met-enkephalin-Arg-Gly-Leu: prohormone analogs that retain good potency and selectivity for δ opioid receptors

We report here on the binding affinity and bioassay results of cyclic enkephalin analogs comprising a cyclic moiety and C-terminal fragment of MERGL, where ME denotes methionine enkephalin. MERGL (YGGFMRGL) has been suggested to be cleaved enzymatically by membrane-bound enkephalinase 24.11 to leave ME and the tripeptide RGL. In our study we have synthesized hybrids of DPDPE or DPLCE and the C-terminal tripeptide RGL in order to mimic a prohormone able to cross the blood–brain barrier. The study has shown that of the homologs presented here, analogs of DPLCE often are more potent at delta opioid receptors both in binding affinity and in bioactivity at the MVD, than DPDPE. Our hypothesis that hybrids (consisting of the drug and the spacer for the carrier) could be designed which would either have no opioid activity or, alternatively, be by themselves very active, has been verified.     

Distinct conformational preferences of three cyclic β-casomorphin-5 analogs determined using NMR spectroscopy and theoretical analysis

The conformational properties of three cyclic β-casomorphin analogs based on the general formula H-Tyr-c[-D-Orn-2-Nal-D-Pro-Xaa-] (2-Nal = 2-naphthylalanine; Xaa = D-Ala, Sar or NMe-Ala) in DMSO solution were investigated using NMR spectroscopy in conjunction with molecular modeling techniques. The D-Ala⁵- and Sar⁵-analogs (compounds 1 and 2, respectively) are potent mixed μ-agonist/§-antagonists with high μ- and §-opioid receptor affinities, whereas the NMe-Ala⁵-analog (compound 3) is a potent μ-agonist and a weak partial §-agonist. Distinct conformational differences emerged for the three compounds studied. Flexibility in the bare ring structures was found to increase in the order 3<2<1. The increased structural rigidity of 3 may be responsible for its low §-receptor affinity as compared to the two other analogs. A low fractional population of conformers containing two cis peptide bonds was found for compound 2 but not for analog 1 or 3. Initial evidence for this observation was obtained from NMR differential line-broadening experiments and later confirmed by molecular mechanics simulations. Comparison of the temperature dependence of amide proton chemical shifts acquired for the three cyclic analogs indicate a large degree of intramolecular hydrogen bonding for 1 but not for the other two peptides. © Munksgaard 1996.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a μ selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a δ selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO₂) or Nip, ii) an azido group as Phe(4 N₃) or AZ. Pharmacological responses on mouse vas deferens (δ sites) and guinea pig ileum (μ sites), as well as competition experiments with [³H] DAGO and [³H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the μ sites, NipDTLET for the δ ones. However the nitro probes do not appear to be photo-activable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the δ site.     

Effect of aromatic amino acid substitutions in the 3-position of cyclic β-casomorphin analogues on μ-opioid agonist/δ-opioid antagonist properties*

The β-casomorphin-5 analog H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] (2-Nal = 2-naphthylalanine) was the first reported cyclic opioid peptide with mixed μ agonist/δ antagonist properties [R. Schmidt et al. (1994) J. Med. Chem. 37 , 1136-1144]. The 2-Na1³ residue in this peptide was replaced with benzothienylalanine (Bta) (3), His(Bz1) (4), Tyr(Bz1) (5), 4′-benzoylphenylalanine (Bpa) (6), 4′-benzylphenylalanine (Bzp) (7), thyrnine (Thy) (8), thyroxine (Thx) (9), 4′-biphenylalanine (Bip) (10), 4′-biphenylglycine (Bpg) (12) and 3,3-diphenylalanine (Dip) (14), and the in vitro opioid activity profiles of the resulting compounds were determined in μ and δ receptor-representative binding assays and bioassays. Analogues 3, 12 and 14 were full agonists in the μ receptor-representative guinea-pig ileum (GPI) assay and also were agonists in the δ receptor-representative mouse vas deferens (MVD) assay. The agonist effects of the latter compounds in the MVD assay were antagonized by the highly selective δ antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), indicating that they were triggered by δ receptor activation. The Bzp³- and Bip³-containing peptides 7 and 10 turned out to be μ antagonists against the μ selective agonist H-Tyr-D-Ala-Phe-Phe-NH₂, in the GPI assay. The other analogues were weak partial μ agonists which displayed remarkably decreased μ receptor affinity as compared to parent peptide 1. Compounds 4-10 were found to be δ antagonists in the MVD assay. Analogues 4 and 9 exhibited δ antagonist potency similar to that of parent peptide 1, while compounds 5-8 and 10 showed 3-12-fold higher δ antagonist potency against DPDPE and deltorphin I and, in most cases, increased δ receptor affinity. These results indicate that the & delta; receptor tolerates bulky aromatic side chains in the 3-position of cyclic β-casomorphin analogs with either δ agonist or δ antagonist properties. However, these compounds displayed drastically reduced μ receptor affinity in nearly all cases. © Munksgaard 1996.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

Nuclear expression of μ‐opioid receptors in a human mesothelial cell line

1 Possibly acting via μ‐opioid receptors (MORs), morphine inhibits the formation of experimentally induced postoperative abdominal adhesions in rats. Mesothelial cells may participate in adhesion formation by secreting mediators that interfere negatively with fibrinolysis. Morphine may prevent adhesions by inhibiting the release of pro‐adhesion mediators from mesothelial cells. This study aimed to investigate whether human mesothelial cells express ΜΟR‐1; if so, such could constitute a site of action for morphine in adhesion prevention. 2 Cells from Met‐5A, a human mesothelial cell line were seeded and prepared for immunocytochemistry and Western blotting. 3 Immunocytochemistry showed MOR‐1 expression in mesothelial cells, predominantly in the nuclei. Western blotting showed two bands (c. 35 and 50 kDa) which correspond to those obtained with a control lysate from cells known to express MORs. In addition, we found MOR‐1 expression with nuclear and cytoplasmatic localization in biopsies from human abdominal adhesions. 4 The current findings may suggest that morphine could interact directly with mesothelial cells via MOR‐1 receptors, and thereby modulate adhesion formation, possibly by interfering with the release of pro‐adhesion factors from these cells.     

Novel μ-selective Met-enkephalinamide analogs with antagonist activity.

Four novel μ-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, β-methyl-m-Tyr, N-phenethyl-m-Tyr and α, β-dimethyl-m-Tyr into D-Ala²-Met⁵-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala²-Met⁵-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a β-CH₃ group to the m-Tyr enhances μ-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high μ-receptor affinity examined have a common energy accessible B'_II 2–3 turn conformation similar to that previously identified for high μ-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.     

Fluorescence study on the solution conformation of dynorphin in comparison to enkephalin

Conformational parameters of the opioid peptides dynorphin and [Leu⁵] enkephalin in dilute aqueous solution (3 times 10^-5 M) were investigated by performing singlet-singlet energy transfer experiments with dynorphin and with the biologically active 4-tryptophan analogs of dynorphin-(1–13) and [Leu⁵] enkephalin at pH 5.5 and 8.0. Efficiencies of transfer of excitation energy from the phenol ring of tyrosine (donor) to the indole moiety of tryptophan (acceptor) were determined and average intramolecular Tyr-Trp distances were calculated on the basis of the Förster equation. The observed absence of energy transfer between Tyr¹ and Trp¹⁴ of dynorphin indicates that the two fluorophores are at least 20 Å apart and rules out a close proximity between the N- and C-terminal segments of the peptide. Evaluation of energy transfer in [Trp⁴] dynorphin-(1–13) resulted in an average intramolecular Tyr¹-Trp⁴ distance of at least 15 Å whereas the corresponding average distance in [Trp⁴, Leu⁵] enkephalin was found to be much shorter (10 Å). It thus appears that in [Trp⁴] dynorphin-(1–13) the predominant conformation of the N-terminal tetrapeptide segment is almost completely extended, whereas in [Trp⁴, Leu⁵] enkephalin folded conformations of that same segment occur in a major proportion. This drastic conformational difference is of interest with regard to the different preferences of dynorphin and [Leu⁵] enkephalin for the various opiate receptor subclasses.