Mouse Vas Deferens Assay Research Articles

Pseudopeptide analogs of substance P and leucine enkephalinamide containing the .psi.(methyleneoxy) modification: synthesis and biological activity

The isosteric methyleneoxy psi (CH2O) function was employed as a novel peptide-bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu6,Phe8 psi(CH2O)Gly9]SP6-11 (7) of SP related C-terminal hexapeptide [pGlu6]SP6-11 and two pseudopeptide analogues of [Leu5]enkephalinamide, [Tyr1 psi (CH2O)Gly2, Leu5] enkephalinamide (11) and [Gly2 psi (CH2O)-Gly3, Leu5]enkephalinamide (17), were synthesized. The N alpha-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC50 = 4.8 nM) of substance P as compared to the parent peptide [pGlu6]SP6-11 (EC50 = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC50 (NK-1)/EC50 (NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu6]SP6-11, EC50 (NK-1)/EC50 (NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC50 = 2 microM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC50 = 2.3 microM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the mu and delta binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC50 = 30 nM), being equipotent to [D-Ala2, Leu5]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC50 = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both mu and delta binding sites with increased selectivity for the delta sites as shown by the ratio of the apparent affinities for both receptors, Ki (delta)/Ki (mu) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed.

Cyclic] [D-Pen2,D-Pen5]enkephalin analogs with increased affinity and selectivity for .delta.-opioid receptors

The conformationally restricted, cyclic disulfide-containing enkephalin analogue [D-Pen2,D-Pen5]enkephalin (DPDPE) was modified by halogenation (F, Cl, Br, I) of the phenylalanine-4 residue in the para position. The potency and selectivity of these analogues for the delta opioid receptor was greater than that of the parent peptide. The analogues possessed greater potency and affinity for the delta receptors than DPDPE in the mouse vas deferens assay and in radioreceptor assays (against [3H]DPDPE), respectively. [p-ClPhe4]DPDPE was the most selective in the radioligand binding assays (IC50(mu)/IC50(delta) = 574), being about 5-fold more delta opioid receptor selective than DPDPE in this assay, whereas [p-IPhe4]DPDPE was the most selective in the classical bioassay systems using the mouse vas deferens and guinea pig ileum assays (IC50(GPI)/IC50(MVD) = 17,374), making it nearly 9-fold more selective than DPDPE in direct comparisons using the same assay conditions.

Biological activities of cyclic enkephalin pseudopeptides containing thioamides as amide bond replacements

Analogs of H-Tyr-cyclo(N ε-D-Lys-Gly-Phe-Leu) have been prepared which contain thioamides at the 3–4 position (monothio), 3–4 and 5-2 positions (dithio), and 2–3, 3–4, and 5-2 positions (trithio). These compounds have been tested for opioid activity in μ- and δ-receptor selective bio- and binding assays. As the number of sulfurs increased, the biological activities dropped on the guinea pig ileum and fluctuated modestly on the mouse vas deferens assay. Surprisingly, the compounds displayed increasing δ selectivity as the number of sulfurs increased. In the binding assay, the thioamide analogs tended to retain affinity toward the μ receptor. The mono- and dithio-analogs were more μ selective than the parent, while the trithio-analog was more δ selective. These results suggest that the subtle exchange of sulfur for oxygen can have a significant impact on receptor selectivity and affinity, and probably reflect the different conformational/structural requirements for binding vs. the biological transduction event.

Synthesis of highly .mu. and .delta. opioid receptor-selective peptides containing a photoaffinity group

A series of cyclic, conformationally constrained photolabile peptides related to the enkephalins and to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the delta and mu opioid receptors. The following new peptides were prepared and tested for their delta opioid receptor potency and selectivity in the guinea pig ileum assay, the mouse vas deferens assay, and the rat brain binding assay: H-Tyr-D-Pen-Gly-p-NH2Phe-D-Pen-OH (1, [p-NH2Phe4]DPDPE) and H-Tyr-D-Pen-Gly-p-N3Phe-D-Pen-OH (2, [p-N3Phe4]-DPDPE). The following new peptides were prepared and tested for their mu opioid receptor potency and selectivity in the same assays: H-D-Phe-Cys-p-NH2Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (3, [p-NH2Phe3]CTP) and D-Phe-Cys-p-N3Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (4, [p-N3Phe3]CTP). The delta selective photoaffinity peptide 2 displayed both high affinity (IC50 = 9.5 nM) and good selectivity (IC50 mu/IC50 delta = 1053) as an agonist at delta opioid receptors in bioassays, and 2 also displayed moderate affinity (33 nM) and excellent selectivity (IC50 mu/IC50 delta = 110) for rat brain delta opioid receptors. The mu selective photoaffinity peptide 4 displayed very weak affinity (8% contraction at 300 nM) at mu opioid receptors in bioassays, but good affinity (IC50 = 48.6 nM) and excellent selectivity (IC50 delta/IC50 mu = 412) for the rat brain mu opioid receptors. These conformationally constrained cyclic photoaffinity peptides may be useful tools to investigate the pharmacology of delta and mu opioid receptors.

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: Development of a ligard with high delta opioid receptor selectivity and affinity

Tetrapeptides of primary sequence Tyr-X-Phe-YNH 2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both μ and δ opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen 4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen 4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from δ selective to μ selective as the ring size was increased. The relatively high binding affinity and δ receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed δ receptor binding selectivity similar to that of the standard δ ligand, [D-Pen 2, D-Pen 5] enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.

Partial characterization of a novel endogenous opioid in human cerebrospinal fluid

Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated “Peak B” has previously been shown to be related to the pain status of chronic pain patients. We now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (< 1.0 μM) but not at higher (>6.0 μM) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or α-chymotrypsin.

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays.

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.

In vitro activity profiles of cyclic and linear enkephalin pseudopeptide analogs

The peptide bond in the 4–5 position of the cyclic and linear enkephalin analogs H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] and H-Tyr-D-Ala-Gly-Phe-L(or D)-Leu-OH was replaced by a thiomethylene ether linkage. Each of the configurational isomers of the cyclic pseudopeptide H-Tyr-cyclo[-D-Lys-Gly-Pheψ[CH 2S]L(or D)-Leu-] showed high potency in both the guinea pig ileum and the mouse vas deferens assay and, therefore, had no preference for either μ- or δ-opioid receptors, in contrast to the cyclic parent peptides H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] which are μ-receptor selective. The loss of selectivity observed with the cyclic pseudopeptides may be due to the greater flexibility of their 18-membered ring structures as a consequence of the peptide bond substitution. The linear pseudopeptide analogs were both less potent and less δ-receptor selective than their parent compounds. These results indicate that thiomethylene ether peptide bond replacements can have a pronounced effect on the activity profile of peptide hormones and neurotransmitters.

Opioid activity of pro-enkephalin-derived peptides in mouse vas deferens and guinea pig ileum

The inhibitory activity of several pro-enkephalin A-derived opioid peptides, containing the sequence of Met-enkephalin, was evaluated in two isolated organ preparations sensitive to opioids, the guinea pig ileum (GPI) and the mouse vas deferens (MVD). All peptides tested were able to inhibit the electrically stimulated contraction in both tissues by interacting with specific receptors sensitive to the opioid antagonist naloxone. The shorter peptides in this family (Met-enkephalin, Met 5-enkephalin-Arg 6-Phe 7, Met 5-enkephalin-Arg 6-Gly 7-Leu 8) displayed their highest potency in MVD. In contrast, the larger bovine adrenal medulla (BAM) peptides (BAM 12P, BAM 22P and peptide E) were more potent in the GPI system compared with the MVD assay. Therefore, the larger peptides seem to bind better to the μ receptor, whereas the shorter ones prefer the δ type.

Prodynorphin opioid peptides in small somatosensory primary afferents of guinea pig

By the use of light microscopic immunohistochemistry it was shown that a significant population of small primary sensory afferents of guinea pig contains immunoreactivities to antisera directed against prodynorphin-opioid peptides, whereas immunoreactivities to antisera directed against opioid peptides exclusively contained in proenkephalin were absent. Immunoreactivities to antisera against different prodynorphin-opioid peptides were seen in small ganglionic cells and in small diameter fibers of spinal and trigeminal ganglia, of dorsal roots, of somatic peripheral nerve trunks and in cutaneous sensory nerves. There was evidence for colocalization of different prodynorphin-opioid peptides. High-performance liquid chromatography and the mouse vas deferens assay revealed opioid-active material (22.7 pmol Met-enkephalin equivalents per gram wet weight) in extracts of somatic peripheral nerves. The results indicate for the first time that opioid peptides derived from prodynorphin are peripheral and central neurotransmitter candidates of small primary somatosensory afferents.

A novel cyclic opioid peptide analog showing high preference for μ-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.

Proenkephalin- and prodynorphin- derived opioid peptides in guinea-pig heart

consecutive high performance liquid chromatography (HPLC) fractionation and the mouse vas deferens assay were used to characterize opioid peptides in the guinea-pig heart. Atria were found to contain at least nine different opioid peptides derived from proenkephalin and prodynorphin. In ventricles at least seven different molecular species which may be derived only from prodynorphin were present. The total opioid activity in atria averaged 22 pmol and in ventricles 11 pmol [Met]enkephalin equivalents per g wet wt. The high content of [Leu]enkephalin in relation to [Met]enkephalin indicates the possibility of a dynorphinergic pathway of cardiac [Leu]enkephalin. Multiple cardiac opioid ligands and receptors, including kappa, may be functionally important in the peripheral control of cardiac performance and coronary circulation.

Receptor binding and biological activity of bivalent enkephalins

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH 2) n - X, where X is H-Tyr- gd-Ala-Gly-Phe-Leu-NH-( n = 0, 2, 4, 6, 8, 10,12), for the first series of dimeric pentapeptide enkephalins (DPE n ), and H-Tyr- d-Ala-Gly-Phe-NH-( n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTE n ). Comparison of biological activities with bincling affinities revealed that: (1) the DPE series with n = 2–8 showed increased potency in the MVD assay relative to monomeric [ d-Ala 2, Leu 5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay than predicted on the basis of bincling affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2–12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta bincling affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponcling monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and μ-bincling but not with MVD and δ-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and bincling assays. Comparison of these compounds with the corresponcling dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.

Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors.

The conformationally restricted, cyclic, disulfide-containing, enkephalin analogs [2-D-penicillamine, 5-L-penicillamine]enkephalin [(D-Pen2,L-Pen5]enkephalin) and [2-D-penicillamine, 5-D-penicillamine]enkephalin [(D-Pen2,D-Pen5]enkephalin) were synthesized by solid-phase methods. Selectivities of these analogs for a single class of opioid receptor were investigated by examining relative potencies in the mouse vas deferens assay, in which the functional receptor is the delta receptor, versus the guinea pig ileum assay, in which the mu receptor is the functional receptor, and by determining their relative abilities to displace the prototypical delta receptor ligand [D-Ala2, D-Leu5]enkephalin and the prototypical mu receptor ligand naloxone from rat brain membrane preparations. Based on these comparisons [D-Pen2,L-Pen5]- and [D-Pen2,D-Pen5]enkephalin exhibited delta receptor selectivities of 1,088 and 3,164, respectively, in the bioassays, and 371 and 175, respectively, in the binding assays. Compared with the previously reported delta receptor selective analogs, [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Thr2,Leu5,Thr6]enkephalin, the bis-Pen-containing analogs provide an order of magnitude increase in delta receptor selectivity.

Cyclic and acyclic partial retro-inverso enkephalinamides: Mu receptor selective enzyme resistant analogs

The cyclic partial retro-inverso modified enkephalins, H-Tyr-cyclo[-D-Glu-Gly-gPhe-D-Leu-] (I), H-Tyr-cyclo[-D-A2bu-Gly-gPhe-R&S-mLeu-] (IIf, IIs), H-Tyr-cyclo[-D-Glu-Gly-Phe-gLeu-] (III), and their corresponding acyclic analogs, H-Tyr-D-Ala-Gly-gPhe-D-Leu-For (Ia), H-Tyr-D-Ala-Gly-gPhe-R,S-mLeu-NH2 (IIa), H-Tyr-D-Ala-Gly-Phe-gLeu-For (IIIa) have been evaluated in in vitro bioassays. Relative to Leu-enkephalin, the modified compounds are more potent in the guinea pig ileum assay and more effectively displace [3H]naloxone from rat brain receptors, while they are generally less potent in the mouse vas deferens assay and only weakly able to displace [3H] (D-Ala2, D-Leu5)enkephalin. The analogs are highly resistant to proteolytic degradation by rat brain membrane preparations that readily hydrolyze Leu-enkephalin.

ChemInform Abstract: SYNTHESIS AND PHARMACOLOGICAL CHARACTERIZATION IN VITRO OF CYCLIC ENKEPHALIN ANALOGS: EFFECT OF CONFORMATIONAL CONSTRAINTS ON OPIATE RECEPTOR SELECTIVITY

AbstractGemäß Formelschema werden die Verbindungen (IV) dargestellt.

Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity

The enkephalin analogs, [ D -Pen 2, L -Cys 5]- and [ D -Pen 2, D -Cys 5]- enkephalin are cyclic compounds, conformationally constrained by virtue of their 14-membered, disulfide containing rings and by the rigidizing effect of the β ,β dimethyl substituents of the penicilamine side chain. The analogs exhibit profound δ receptor specificity as assessed by their relative potencies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, exhibiting, respectively, 666 and 215 times higher potency in the latter assat system. By contrast, the receptor selectivities measured in rat brain binding assays in the absence of sodium were much more modest, the cyclic analogs being, respectively, 15.2 and 6.0 times more effective at displacing [ 3H] [ D -Ala 2, D -Leu 5] enkephalin than [ 3H] naloxone. However, for binding assays performed in the presence of a sodium concentration equivalent to that used in the GPI and MVD assays, these binding selectivities increased to 167 and 49, respectively.

Lysine substitution increases mu-selectivity of potent di- and tri-peptide enkephalin analogs

A general trend toward increased mu-selectivity has been reported in two classes of minimal structure enkephalin analogs which contain the essential structural and functional information for potent enkephalin-like biological activity. A single Lys residue added to the amino terminal of several tripeptide amides, and to the potent dipeptide amide Tyr-DAla-phenylpropylamide, causes a further increase in mu-selectivity as determined in the stimulated guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, and in receptor binding studies in homogenates of guinea pig brain. The effect of Lys substitution was a significant decrease in the delta-related MVD potencies.

Comparison of μ-, δ- and κ-receptor binding sites through pharmacologic evaluation of p-nitrophenylalanine analogs of opioid peptides

For the purpose of comparing the structural requirements of opioid receptor subsites interacting with phenylalanine residues of opioid peptides, analogs containing p-nitrophenylalanine (Phe (pNO 2)) were synthesized and tested in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in μ- and δ-receptor selective binding assays. Whereas substitution of Phe(pNO 2) in position 4 of the μ-selective analog H-Tyr-c[-N ϵ-D-Lys-Gly-Phe-Leu-] and of the δ-selective analog H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH produced a significant potency increase in all assays, the corresponding substitution in the κ-selective dynorphin-(1–13) fragment resulted in a decreased potency in the GPI-assay. These findings suggest that the electronic requirements of the Phe 4 binding site at the μ- and δ-receptor are identical, but different from those of the corresponding site at the κ-receptor. The observation that Phe(pNO 2) substitution in position 3 of morphiceptin and dermorphin also produces a drastic potency drop indicates that the Phe 3 side-chain of the latter peptides at the μ- and δ-receptor may bind to a site different from that interacting with the Phe 4 side-chain of enkephalins.

Synthesis and pharmacological characterization in vitro of cyclic enkephalin analogues: effect of conformational constraints on opiate receptor selectivity.

Using a combination of solid-phase and solution methods, we synthesized a series of cyclic [Leu5]enkephalin analogues by substitution of D-alpha, omega-diamino acids in position 2 of the enkephalin sequence and cyclization of the omega-amino group to the C-terminal carboxy group of leucine. Cyclic analogues containing D-alpha, beta-diaminopropionic acid (1), D-alpha, gamma-diaminobutyric acid (2), D-ornithine (3), or D-lysine (4) in position 2 and the [D-Leu5] and [des-Leu5] analogues of 4 (5 and 6) showed, in general, high potency in the guinea pig ileum (GPI) assay and low potency in the mouse vas deferens (MVD) assay. IC50 (MVD)/IC50 (GPI) ratios ranging from 3.1 to 29.4 were obtained, indicating the preference of the cyclic analogues for mu receptors over delta receptors. With two exceptions, preferential affinity for mu receptors is reflected in the Ki ratios determined in parallel binding assays using [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin as mu and delta receptor selective radioligands, respectively. Comparison of the pharmacological profiles of the cyclic analogues 1-4 with those of their corresponding open-chain analogues, [D-Ala2, Leu5]enkephalinamide (1a), [D-Abu2, Leu5]enkephalinamide (2a), [D-Nva2, Leu5]enkephalinamide (3a), and [D-Nle2, Leu5]enkephalinamide (4a), revealed that the pronounced mu character of compounds 1-4 is a direct consequence of the conformational constraints introduced by cyclization. This finding is in agreement with the concept of different conformational requirements of mu- and delta-opiate receptors and raises the possibility of manipulating opiate receptor selectivity by varying the type and degree of conformational restriction.