Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen2,D-Pen5]enkephalin with delta opioid receptor selectivity.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5] enkephalin (DPDPE) was modified by 2' (CH3) and 3' (I, OCH3, NO2, NH2) ring substitutions and by beta-methyl conformationally constrained beta-methyltyrosine derivatives in the 1 position. The potency and selectivity of these analogues were evaluated by bioassay in the mouse vas deference (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay) assays and by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H][p-ClPhe4]DPDPE (delta ligand). The analogues showed highly variable potencies in the binding assays and in the bioassays. Aromatic ring substituents with positive Hammett constants had decreased potency, while substituents with negative Hammett constraints has increased potency for the opioid receptor. The most potent and most selective compound based on the binding was [2'-MeTyr1]DPDPE (IC50 = 0.89 nM and selectivity ratio 1310 in the binding assays). The 6-hydroxy-2-aminotetralin-2-carboxylic acid-containing analogue, [Hat1]DPDPE, also was highly potent and selective in both assays, demonstrating that significant modifications of tyrosine in enkephalins are possible with maintenance of high potency and delta opioid receptor selectivity. Of the beta-methyl-substituted Tyr1 analogues, [(2S,3R)-beta-MeTyr1]DPDPE was the most potent and the delta receptor selective. The results with substitution of beta-MeTyr or Hat instead of Tyr also demonstrate that topographical modification in a conformationally restricted ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity.     

Chronic effect of [D-Pen2,D-Pen5]enkephalin on rat brain opioid receptors.

In previous studies, we have demonstrated that chronic etorphine or [D-Ala2,D-Leu5]enkephalin (DADLE) treatment of rats results in the reduction of mu- and delta-opioid receptor binding activities as tolerance develops. As both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu- and delta-receptors, these studies could not determine whether down-regulation of a specific receptor type occurs. Therefore, in the present studies, animals were rendered tolerant to the delta-opioid receptor-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and receptor binding activities were measured. Treating Sprague-Dawley rats with increasing doses of DPDPE (80-160-240-320 micrograms/kg) i.c.v. for 1 to 4 days resulted in a time-dependent increase in the AD50 of DPDPE to elicit an antinociceptive response. When delta-receptor binding was determined by using [3H]DPDPE, a 40-50% decrease in binding in the midbrain and cortex, and 25-35% decrease in binding in the striatum were observed after 3 or 4 days of DPDPE treatment. Scatchard analysis of the [3H]DPDPE saturation binding data revealed a decrease in Bmax values and no significant change in Kd values. To our surprise, when mu-receptor binding was determined by using [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), a 10-15% decrease in binding was also observed in the midbrain and cortex after 4 days of DPDPE treatment. Our conclusion is that chronic DPDPE treatment preferentially reduces delta-opioid receptor binding activity. Its minor effect on the mu-opioid receptor maybe due to an interaction between delta cx and mu cx binding sites.     

Analgesic and tolerance-inducing effects of the highly selective delta opioid agonist [D-Pen2,D-Pen5]enkephalin in mice

The novel and highly selective, conformationally restricted enkephalin analogue for delta-opioid receptors, [D-Pen2,D-Pen5]enkephalin (DPDPE; Pen = penicillamine), was studied in various in vivo tests for analgesia, tolerance and physical dependence. Intracerebroventricular (i.c.v.) administration of DPDPE caused a dose-dependent, naloxone-reversible antinociception, measured with the heat-irradiant (tail-flick) method. Acute tolerance developed to the antinociceptive effect of DPDPE. DPDPE also caused mild signs of physical dependence (withdrawal hypothermia and body weight loss) after repeated peptide treatment. Severe signs of morphine withdrawal (e.g. withdrawal jumping) on the other hand, could not be reversed by the administration of DPDPE. It is concluded that the activation of central delta-opioid receptors may play a role in controlling pain mechanisms, and that this activation is followed by the rapid development of a tolerance to this action.     

Topographically designed analogues of [D-Pen,D-Pen5]enkephalin.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5]enkephalin (1, DPDPE) was systematically modified topographically by addition of a methyl group at either the pro-S or pro-R position of the beta carbon of an L-Phe4 or D-Phe4 residue to give [(2S,3S)-beta-MePhe4]DPDPE (2), [(2R,3R)-beta-MePhe4]DPDPE (3), [(2S,3R)-beta-MePhe4]DPDPE (4), and [(2R,3S)-beta-MePhe4]DPDPE (5). The four corresponding isomers were prepared in which the beta-methylphenylalanine residue was p-nitro substituted, that is with a beta-methyl-p-nitrophenylalanine (beta-Me-p-NO2Phe) residue, to give [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (6), [(2R,3R)-beta-Me-p-NO2Phe4]DPDPE (7), [(2S,3R)-beta-Me-p-NO2Phe4] DPDPE (8), and [(2R,3S)-beta-Me-p-NO2Phe4]DPDPE (9), respectively. The potency and selectivity (delta vs mu opioid receptor) were evaluated by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H]DPDPE (delta ligand) and by bioassay with mouse vas deferens (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay). The eight analogues of DPDPE showed highly variable binding and bioassay activities particularly at the delta opioid receptor (4 orders of magnitude), but also at the mu opioid receptor, which led to large differences (3 orders of magnitude) in receptor selectivity. For example, [(2S,3S)-beta-MePhe4]DPDPE (2) is 1800-fold selective in binding to the delta vs mu receptor, making it one of the most selective delta opioid receptor ligands in the enkephalin series as assessed by the rat brain binding assay, whereas the corresponding (2R,3R)-beta-Me-p-NO2Phe-containing analogue 9 is only 4.5-fold selective (nonselective) in this same assay. On the other hand, in the bioassay systems, [(2S,3S)-beta-Me-p-NO2Phe4]DPDPE (5) is more potent than DPDPE and 8800-fold selective for the MVD (delta receptor) vs the GPI (mu receptor), making it the most highly selective ligand in this series for the delta opioid receptor on the basis of these bioassays. In these assay systems, the (2R,3S)-beta-MePhe4-containing analogue 5 had very weak potency and virtually no receptor selectivity (4.4-fold). These results demonstrate that topographical modification alone in a conformationally restricted peptide ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity and suggest that this approach may have general application to peptide ligand design.     

Theoretical study of the flexibility and solution conformation of the cyclic opioid peptides [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin

An investigation of the conformational profiles of two cyclic delta-selective opioid peptides, [D-Pen2,D-Pen5]-enkephalin and [D-Pen2,L-Pen5]-enkephalin, has been made. The methods and procedures used are more extensive and systematic than those previously reported, involving a combination of nested grid rotations, cyclic ring-closing algorithms, molecular dynamic simulations at high and low temperature, and total geometry optimizations. The reexamination is a necessary first step in further characterization of the bioactive form of delta-selective peptides. This study also addresses the question of how rigid such cyclic analogs actually are. Finally, the effect of solvent environment on the low energy conformers obtained from the extensive search strategy has been determined. Simulation of the effect of water as a solvent by a continuum dielectric constant of 80 results in the breaking of internal hydrogen bonds and rearrangement of the rank order of energy of the conformers. The lowest energy solution conformation for [D-Pen2,D-Pen5]-enkephalin, obtained without utilizing any experimental data, is in excellent agreement with the geometric properties deduced from its solution NMR spectra.     

Systemic analgesic activity and delta-opioid selectivity in [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin.

The cyclic peptide [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin (2) was synthesized by solid-phase techniques and contains the optically pure unnatural amino acid 2,6-dimethyltyrosine (DMT) as a replacement for the Tyr1 residue of [D-Pen2,D-Pen5]enkephalin (DPDPE, 1). This structural modification resulted in a 10-fold increase in the potency of 2 at the delta opioid receptor and a 35-fold increase in potency at the mu receptor while substantial delta receptor selectivity was maintained. In addition, 2 was 86-fold more effective than 1 at inhibiting electrically stimulated contractions of the mouse vas deferens. In the hot plate test, 2 was 7-fold more potent than 1 after intracerebroventricular administration in the mouse. While 1 was inactive following systemic administration of doses as high as 30 mg/kg, subcutaneous administration of 2 significantly inhibited writhing with an ED50 of 2.6 mg/kg. These results demonstrate that the potency and systemic activity of DPDPE are significantly increased by replacement of Tyr1 with DMT.     

Development of novel enkephalin analogues that have enhanced opioid activities at both mu and delta opioid receptors

Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve therapeutic benefit for the treatment of pain due to mixed mu and delta opioid agonist activities. Ligand 16, in which a Dmt-substituted enkephalin-like structure was linked to the N-phenyl-N-piperidin-4-yl propionamide moiety, showed very high binding affinities (0.4 nM) at mu and delta receptors with an increased hydrophobicity (aLogP = 2.96). This novel lead compound was found to have very potent agonist activities in MVD (1.8 nM) and GPI (8.5 nM) assays.     

Modulation of mu-mediated antinociception by delta agonists in the mouse: selective potentiation of morphine and normorphine by [D-Pen2,D-Pen5]enkephalin

The effect of the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) on the antinociception produced by intracerebroventricular (i.c.v.) administration of the mu agonists morphine, [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO), [NMePhe3,D-Pro4]morphiceptin (PLO17), beta-endorphin, phenazocine, etorphine and sufentanil was studied in mice. Only the antinociceptive effects of morphine and normorphine were modulated by i.c.v. coadministration of a dose of DPDPE which did not produce any significant antinociception alone. Both the morphine and normorphine dose-response lines were displaced to the left in the presence of DPDPE. The delta-selective antagonist ICI174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH) (where Aib is alpha-aminoisobutyric acid) blocked the modulation of morphine antinociception by DPDPE. ICI 174,864 alone failed to produce either a significant increase or decrease of morphine, phenazocine, etorphine or beta-endorphin antinociception. The results of the present study provide support for the hypothesis that the enkephalins may function to modulate antinociception produced at the mu receptor; such modulation may come about via the existence of an opioid mu-delta receptor complex. The mu receptors existing in such a complex may be selectively activated by morphine and normorphine, but not the other mu agonists studied here. Thus, the enkephalins may function both to directly initiate, as well as to modulate, some forms of supraspinal mu receptor-mediated antinociception.     

Effects of [D-Ala2, D-Leu5]enkephalin and [D-Pen2, L-Pen5]enkephalin on apomorphine-induced motor activity in the mouse.

The effects of intracerebroventricular injections of opioid peptides such as DADL [( D-Ala2, D-Leu5]enkephalin) and DPLPE [( D-Pen2, L-Pen5]enkephalin) with different degrees of selectivity for delta- over mu-receptor on apomorphine (0.1, 0.3, 1.0 and/or 3.0 mg/kg)-induced motor activity were investigated in the mouse using multi-dimensional behavioral analyses. Lower doses (0.1 and 0.3 mg/kg) of apomorphine failed to affect significantly motor activity, whilst higher doses (1.0 and 3.0 mg/kg) of the drug produced a marked increase in linear locomotion, circling, rearing, and/or grooming behaviors. DADL (0.03, 0.1 or 0.3 microgram) by itself did not influence behaviors, while the peptide (0.1 and 0.3 microgram) produced a marked inhibition on apomorphine (1.0 but not 3.0 mg/kg)-induced increase in rearing behaviors. Furthermore, the inhibitory effect of DADL (0.3 micrograms) on the apomorphine (1.0 mg/kg)-induced increase in rearing was reversed by treatment with the alkylating agent beta-FNA (beta-funaltrexamine) (5.0 micrograms). In contrast to the effects of DADL, the much more delta-selective opioid agonist DPLPE (0.3, 1.0 or 1.75 micrograms) had no marked effects on apomorphine (1.0 mg/kg)-induced behaviors. These results suggest that delta opioid receptors do not play a principal role in the apomorphine-induced increase in circling, rearing or grooming behaviors.     

Development of conformationally constrained linear peptides exhibiting a high affinity and pronounced selectivity for delta opioid receptors

A series of linear conformationally constrained opioid peptides was designed in an attempt to develop highly selective and potent agonists for the delta opioid receptors. These enkephalin analogues corresponding to the general formula Tyr-D-X(OY)-Gly-Phe-Leu-Thr(OZ) were obtained by incorporating bulky residues (X = Ser or Thr; Y = tert-butyl or benzyl; Z = tert-butyl) into the sequence of the previously reported delta specific agonists DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr). In binding studies based on displacement of mu and delta opioid receptor selective radiolabeled ligands from rat brain membranes, the two constrained hexapeptides, Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr (1, DSTBULET) (KI(mu) = 374 nM, Kr(delta) = 6.14 nM, KI(delta)/KI(mu) = 0.016) and in particular Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr(O-t-Bu) (7, BUBU) (KI(mu) = 475 nM, KI(delta) = 4.68 nM, KI(delta)/KI(mu) = 0.010) were shown to be among the most potent and selective delta probes reported to date. A roughly similar pattern of selectivity was obtained with the guinea pig ileum and mouse vas deferens bioassays. In addition, the analgesic potency (hot-plate test) of these peptides intracerebroventricularly administered in mice was shown to be significantly related to their mu-receptor affinity.     

Lipid membrane permeability of modified c[D-Pen2, D-pen5]enkephalin peptides

Permeability coefficients of a series of analogues of a potent opioid peptide, c[D-Pen², D-Pen⁵]enkephalin, were measured in a model membrane system. The analogues included hydrophobic amino acid substitutions on position 3. Liposomes of a mixed composition consisting of zwitterionic lipids and cholesterol served as the model membranes. The obtained permeability coefficients range between 0.38 × 10^?12 and 2.9 × l0^?12 cm/s. These data were correlated with the hydrophobicity scale of Nozaki and Tanford (J. Biol. Chem. 246, 1971, 2211-2217) (correlation coefficient = 0.9933) and with determinations of lipid order perturbation by differential scanning calorimetry (correlation coefficient = -0.9779). The reasonably good correlation obtained within the family of analogues substituted on position 3 (Gly, Ala, Leu, Phe) indicates that changes in permeabilities are primarily related to increases in the partition coefficient of the peptide. However, Phe residue added on the N-terminal end of the peptide (position 0) does not appear to follow the observed trend, showing stronger lipid perturbation and lower permeability compared to the Phe³ analog. This observation demonstrates that each class of peptide modifications requires a new basis of permeability analysis and predictions. © Munksgaard 1996.     

Cholecystokinin analogues with high affinity and selectivity for brain membrane receptors*

A series of CCK analogues in which positions 28 and 31 have been replaced by N-methylnorleucine residues have been synthesized. It has been found that most of these N-methylnorleucine containing analogues of CCK are highly potent and some are extraordinarily selective for the central vs. peripheral receptor in two animal models (guinea pig and rat). [N-MeNle^28.31] CCK_26–333 nonsulfated exhibited both high potency (IC₅₀= 0.13 nM) and selectivity for central vs. peripheral receptors. The pancrease to brain cortex binding affinity ratio for this analogue IS 5100 in the rat model. NMR studies reveal that there is cis/trans isomerism about the N-methylnorleucine residue that may be related to high selectivity.     

Delta-opioid-receptor activation by [D-Pen2,D-Pen5]enkephalin and morphine inhibits substance P release from trigeminal nucleus slices.

The release of substance P (SP) from spinal dorsal horn slices is partially inhibited by micromolar concentrations of selective delta-opioid receptor agonists. In the present study, we have examined the effect of nanomolar concentrations of [D-Pen2,D-Pen5]enkephalin (DPDPE, delta-opioid receptor agonist) and low micromolar of concentrations morphine on K(+)-evoked SP release from rat trigeminal nucleus caudalis (TNC) slices. DPDPE and morphine inhibited SP release with an apparent maximal effect at 3 nM and at 3 microM, respectively. DPDPE and morphine produced U-shaped concentration-response curves that were completely autoinhibited at 100 nM DPDPE and 1 microM morphine. The inhibition of SP release produced by 3 nM DPDPE and 3 microM morphine was blocked by the opioid receptor antagonists naloxone (30 nM; non-selective) and ICI 174,864 (0.3 microM; delta-selective) but not by nor-binaltorphimine (3 nM n-BNI; kappa-selective), naloxonazine (1 nM; micro 1-selective) or beta-funaltrexamine (20 nM beta-FNA; mu-selective). These findings indicate that delta-opioid receptor-mediated inhibition of SP release from TNC can be achieved by nanomolar concentrations of selective delta-opioid receptor agonists. Activation of delta-opioid receptors by morphine might be involved in the residual analgesia observed after mu 1-opioid receptor blockade and in the analgesia produced by high doses of morphine.     

[d-Pen2,d-Pen5]enkephalin,the standard delta opioid agonist,induces morphine-like behaviors in mice

[d-Pen²,d-Pen⁵]enkephalin (DPDPE; 3–30 µg) and morphine (10 µg) both caused Straub tails, increased locomotion, and circling after ICV administration to ICR mice. DPDPE-induced tail stiffening was reduced when mice were pretreated with naloxone (0.5 mg/kg SC) or-funaltrexamine (10 µg ICV), but not with ICI 174864 (2 mg/kg SC), the selective antagonist at delta opioid receptors. These results point to (a) mu receptors mediating the tail stiffening and (b) the loss of delta receptor selectivity after 10 and 30 µg DPDPE.     

The use of [3H]-[D-Pen2,D-Pen5]enkephalin as a highly selective ligand for the delta-binding site.

The characteristics of the binding of [3H]-[D-Pen2,D-Pen5]enkephalin were determined in homogenates of guinea-pig and rat brain. In the guinea-pig, the maximum binding capacity for [3H]-[D-Pen2,D-Pen5]enkephalin was 4.19 pmol g-1 and the KD 1.61 nM. In the rat, the corresponding values were 2.47 pmol g-1 and 5.42 nM. In both species, the maximum binding capacity and the affinity were not altered when mu-binding was suppressed with [D-Ala2,MePhe4,Gly-ol5]enkephalin. The mu-agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin and morphine, displaced a small portion of the binding of [3H]-[D-Pen2,D-Pen5]enkephalin with high affinities.     

Biologically active conformation of [Met5]- and [Leu5]enkephalin on delta opioid receptor

Investigations of the conformation of endogenous enkephalins are generally based on structural comparisons of enkephalins with other opiates and on experimental pharmacological studies. Based on such investigations, we now propose a novel model of the biologically active (receptor-bound) conformation of [Met5]- and [Leu5]enkephalin on the delta opioid receptor. The model helps with the design of new opioid analgesics.     

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.     

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.     

D-Ala2,NMePhe4,Gly-ol5]enkephalin, but not [D-Pen2,L-Pen5]enkephalin, specifically inhibits behaviors induced by the dopamine D2 agonist RU 24213.

The effects of intracerebroventricular injection (10 microliters) of mu- and delta-selective opioid peptides on behaviors induced by the dopamine D2-selective agonist RU 24213 were investigated in the mouse, using multi-dimensional behavioral analyses. Fifteen to 30 min after the start of behavioral measurements, a 3.0 mg/kg dose of RU 24213 produced a marked increase in linear locomotion, circling, rearing and grooming behaviors. Although the mu-selective opioid peptide [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) (0.003 and 0.01 microgram) itself did not significantly affect behaviors, DAGO (0.01 microgram) antagonized the RU 24213 (3.0 mg/kg)-induced increase in behaviors such as linear locomotion, circling, rearing, and grooming. Additionally, the effects of DAGO on RU 24213-induced behaviors were fully reversed by treatment with the mu-selective alkylating agent beta-funaltrexamine (beta-FNA) (5.0 micrograms). In contrast, the delta-selective opioid peptide [D-Pen2,L-Pen5]enkephalin (0.3 or 1.0 micrograms) had no marked effects on RU 24213 (3.0 mg/kg)-induced behaviors. These results suggest that mu- but not delta-opioid receptors play an inhibitory role in the behaviors induced by the selective activation of dopamine D2 receptors.