Tetrapeptide derivatives of [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) lacking an N-terminal tyrosine residue are agonists at the mu-opioid receptor.

The Phe(1) cyclic tetrapeptide Phe-c[D-Cys-Phe-D-Pen]NH(2) (Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the mu-opioid receptor. To examine the role of the Phe(1) residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [(35)S]GTPgammaS assay. Alteration of the bridging groups between the D-Cys(2) and D-Pen(4) residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining mu- and delta-affinities. The one carbon distance between the alpha carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe(1) residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3, 3-(diphenyl)alanine. Conformational restriction of the Calpha-Cbeta and/or Cbeta-Cgamma bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline(1)-containing peptides. Most surprisingly, replacement of the Phe(1) aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (K(i) = 32.5 nM) and potency (EC(50) = 58.8 nM). Thus, the tyrosyl para-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for mu-opioid receptor affinity, agonist potency, and efficacy.

kappa-Opioid receptor binding populations in rhesus monkey brain: relationship to an assay of thermal antinociception.

The binding characteristics of the kappa opioid ligands [3H]U69,593 and [3H]bremazocine, the mu opioid ligand [3H][D-ala2,N-Me-Phe4,glycol5]enkephalin and the delta opioid ligand [3H]p-Cl-[D-pen2,5]enkephalin were studied in rhesus monkey brain membranes in saturation binding experiments and were followed by competition binding experiments with a variety of peptidic and nonpeptidic opioid ligands. The [3H]U69,593 sites appeared to be a subset of kappa opioid receptors (kappa-1 receptors: Kd, 1.2 nM; Bmax, 66 fmol/mg). [3H]Bremazocine (in the presence of mu and delta receptor-masking agents), bound to a larger population of kappa receptors (kappa-all: Kd, 0.39 nM; Bmax, 227 fmol/mg), which presumably included the aforementioned kappa-1 sites. Competition binding experiments revealed that the presently defined kappa-1 sites were similar to previously reported sites in other mammalian species, particularly in terms of the higher kappa-1 selectivity observed with arylacetamide (e.g., U50,488) vs. benzomorphan kappa agonists (e.g., ethylketocyclazocine). The kappa-selective antagonist norbinaltorphimine (nor-BNI) displayed a very small (2.3-fold) selectivity for kappa-1 vs. kappa-all sites. This led to the prediction that in rhesus monkeys (n = 3), systemically administered nor-BNI (10 mg/kg s.c.) should have a very moderate degree of antagonist selectivity for the antinociceptive effects of a putative kappa-1-agonist, the arylacetamide U50,488 (0.1-3.2 mg/kg s.c.), vs. those of the benzomorphan kappa agonist ethylketocyclazocine (0.01-056 mg/kg s.c.). This prediction was confirmed in vivo because nor-BNI (10 mg/kg) caused a robust and long lasting (up to 21 days) antagonism of the antinociceptive effects of U50,488 and a small but significant antagonism of ethylketocyclazocine. The arylacetamide congener Cl-977 (enadoline), which displayed an 11-fold kappa-1 vs. kappa-all binding selectivity, was not sensitive to nor-BNI pretreatment. This indicates that the kappa subtype-binding profile of an agonist is not necessarily predictive of its sensitivity to nor-BNI in vivo. Overall, the present results suggest that at least two functional kappa receptor populations may be present in rhesus monkey brain.

A high affinity, mu-opioid receptor-selective enkephalin analogue lacking an N-terminal tyrosine.

We report a high affinity, mu opioid receptor selective enkephalin analogue in which the N-terminal tyrosine residue thought to be required for such high affinity is replaced by phenylalanine. The high affinity can be traced to a shift of the ligand's N-terminal residue within the mu receptor binding pocket, which diminishes the importance of the usual hydrogen bond between the tyrosine phenolic moiety and the receptor.

Development of a model for the delta opioid receptor pharmacophore. 1. Conformationally restricted Tyr1 replacements in the cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13).

A series of analogues of the conformationally restricted delta opioid receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM 13) was prepared in which the conformationally labile Tyr residue was replaced with several less flexible tyrosine analogues. Among these tyrosine analogues were the bicyclic structures 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (HO-Tic), 2-amino-6-hydroxytetralin-2-carboxylic acid (Hat), and 2-amino-5-hydroxyindan-2-carboxylic acid (Hai) in which rotations about the C alpha-C beta and C beta-C gamma bonds are restricted due to cyclization of the side chain to the backbone. Also examined were analogues in which tyrosine was replaced with either trans-3-(4'-hydroxyphenyl)proline (t-Hpp) or cis-3-(4'-hydroxyphenyl)proline (c-Hpp), residues in which rotations about C alpha-C beta, but not C beta-C gamma, are restricted. Both the t-Hpp1 and c-Hpp1 analogues displayed delta receptor binding affinity similar to the parent Tyr1-containing peptide, while the D-Hat1, L-Hat1, and L-Hai1 analogues exhibited somewhat lower affinity. The results observed for the t-Hpp1 and c-Hpp1 analogues are particularly significant since these two residues have little accessible conformational space in common. Since the binding conformation of residue 1 must be included in this limited conformational intersection, its elucidation is facilitated. Bioassay results from guinea pig ileum and mouse vas deferens preparations are in general agreement with the binding results; however some potency discrepancies are observed. These discrepancies may reflect different selectivities among delta receptor subtypes for the analogues or may represent differing efficacies among these conformationally restricted peptides. The conformational properties of the parent tetrapeptide and the residue 1-modified analogues were studied by molecular mechanics computations. All these peptides share a common rigid tripeptide cycle with a single energetically preferred backbone conformation and three different conformers of the D-Cys, D-Pen disulfide bridge, two of which are observed in the solid state and in aqueous solution, as previously determined from X-ray crystallography and 1H NMR spectroscopy data (Lomize, A; et al. J. Am. Chem. Soc. 1994, 116, 429-436). All the peptides have similar sets of low-energy conformations of their common flexible elements, the Phe3 side chain and the peptide group between the first residue and the rigid tripeptide cycle. However, possible conformations of the first residue differ and depend on the covalent constraints incorporated into the side chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide.

The previously described cyclic, delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH, where Pen, penicillamine, is beta-beta-dimethylcysteine, was modified at residues 2 and 4 by varying combinations of D- and L-Cys and D- and L-Pen, and effects on mu and delta opioid receptor binding affinities and on potency in the mouse vas deferens (MVD) smooth muscle assay were evaluated. A comparison was drawn between consequences of alterations in this series of analogs and those of analogous modifications in the related cyclic pentapeptide series which includes the highly delta receptor-selective [D-Pen2,D-Pen5]enkephalin, DPDPE. Unlike effects observed in the cyclic pentapeptide series, the mu receptor binding affinities of the cyclic tetrapeptides are not dramatically influenced by substitution of Pen for Cys at residue 2. Conversely, while binding of the pentapeptides is only slightly affected by alteration of the chirality of the carboxy-terminal residue, modification of stereochemistry at the carboxy terminus in the tetrapeptides critically alters binding behavior at both mu and delta sites. In contrast with the pentapeptide series, the tetrapeptides appear to be highly dependent upon primary sequence for binding and activity, as only the lead compound binds with high affinity to the delta site. Results suggest that the less flexible cyclic tetrapeptides, lacking the Gly3 residue, display more stringent structural requirements for binding and activity than do the corresponding cyclic pentapeptides.

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features.

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.