The role of backbone conformation in deltorphin II binding: a QSAR study of new analogues modified in the 5-, 6-positions of the address domain.

The delta selectivity of the opioid heptapeptides deltorphin I and II has been attributed to the C-terminal 'address' domain, the hydrophobic Val(5)-Val(6) residues apparently playing a topographical role. We now report the synthesis, opioid binding affinities, and a QSAR study of a series of peptides in which one of the valine side chains was altered. QSAR analyses included previously published models for a binding pocket interaction and an optimum size (Schullery, S.; Mohammedshah, T.; Makhlouf, H.; Marks, E.; Wilenkin, B.; Escobar, S.; Mousigian, C.; Heyl, D. Bioorg. Med. Chem. 1997, 5, 2221), and a new approach for backbone conformational effects using Langevin dynamics simulation (PM3 semi-empirical force field) of an isolated peptide fragment containing the side chain and flanking peptide bonds. No evidence is found of binding pocket interactions or optimum size for either the position-5 or -6 side chain. Rather, delta binding is generally disfavored while mu binding is either unaffected (position-5) or favored (position-6) by larger side chains. The dynamics results provide evidence of similar 'local' conformation roles for the positions 5 and 6 side chains. Specifically, delta binding is favored by side chains that maximize the extension of the backbone, measured as the through-space distance between peptide fragment ends, the angle between lines connecting the alpha-carbon with fragment ends, or the difference between the psi and phi peptide angles.

Binding to delta and mu opioid receptors by deltorphin I/II analogues modified at the Phe3 and Asp4/Glu4 side chains: a report of 32 new analogues and a QSAR study.

The synthesis and binding affinities of 32 X3Gly4 dual-substitution analogues of the natural opioid heptapeptides deltorphin I and II are reported. A multiple regression QSAR analysis was performed using those results along with literature data for the X3Asp4 and Phe3X4 side chain analogues. Fitting to a three-term potential well model with hydrophobic and van der Waals attraction terms and a steric repulsion term indicates that the delta and mu receptor sites for binding the residue three side chain are similar, and that the binding interaction is primarily van der Waals and secondarily hydrophobic. Further analysis indicates that both sites are more constrained with respect to side chain length than width or thickness, and the mu site appears to be somewhat larger. A binding model consistent with these findings pictures the native third residues Phe ring laying on a step notched out of the receptor surface, pointing toward the back (riser) of the step, and sandwiched between the receptor and ligand. However, the binding sites for the residue four side chains are quite different on delta and mu receptors. Binding to the delta site appears to involve both electrostatic attraction (probably to a partial positive charge) and van der Waals attraction, but not necessarily hydrogen bonding, and more constraint with respect to side chain length than width or thickness. In contrast, there is no evidence for any kind of binding attraction between the side chain of residue four and the mu site, which acts more as steric repulsion site, as though the space that is a pocket on the delta receptor is filled in on the mu receptor. A regression model based only on steric repulsion by van der Waals bulk and/or the effective bulk of a hydration layer accounts for over 80% of the residue four related variation in mu affinity.

Opioid receptor binding requirements for the delta-selective peptide deltorphin. I: Phe3 replacement with ring-substituted and heterocyclic amino acids.

In order to assess steric, lipophilic, and electronic influences on opioid binding affinity, analogs of the delta receptor selective peptide deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH2) were prepared in which the residue 3 phenylalanine was replaced with lipophilic fluoro- and methyl-substituted phenylalanines or with the heterocyclic aromatic amino acids 3-(4-thiazolyl)alanine, 3-(2-pyridyl)alanine, 3-(3-pyridyl)alanine, histidine, and 3-(4-thiazolyl)alanine. mu binding was variable, with KiS in excess of 10,000 nM for most analogs, and all of the analogs bound poorly to k receptors. Among the phenyl ring-substituted analogs, those containing the smaller and electron-withdrawing halogens were favored over those with larger, electron-releasing methyl groups, although delta opioid binding affinity was reduced in all cases. The m-fluorophenylalanine analog demonstrated the best delta binding of the group, with a Ki of 4.79 nM. Within the group of heterocyclic analogs, 3-(2-thienyl)alanine proved to be the best modification, displaying a delta receptor Ki of 1.38 nM, while the polar histidine analog suffered the greatest loss in delta binding (Ki = 317). Compounds containing pyridylalanine and thiazolylalanine were intermediate in binding affinity, with delta KiS ranging from 39.5 to 62.4 nM. The major factor influencing the opioid binding of the similar-sized heterocyclic compounds was relative lipophilicity, which outweighed electronic character.

Development of a model for the delta opioid receptor pharmacophore. 2. Conformationally restricted Phe3 replacements in the cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13).

The in vitro pharmacological properties and conformational features of analogs of the delta opioid receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) in which the Phe3 residue was replaced by each of the four stereoisomers of beta-methylphenylalanine (beta-MePhe) were investigated. Both analogs in which the alpha carbon of the Phe3 replacement has L-stereochemistry display high affinity for delta receptors with the (2S,3S)-MePhe3 analog exhibiting approximately 8-fold higher affinity than the (2S,3R)-MePhe3 diastereomer. Surprisingly, one analog with D-stereochemistry in residue 3, the (2R,3R)-MePhe3 analog, also displays high affinity for the delta receptor and is extraordinarily selective for this receptor. All analogs were agonists in the mouse vas deferens (MVD) and guinea pig ileum (GPI) smooth muscle bioassays, displaying MVD and GPI potencies consistent with their delta and mu opioid receptor affinities, respectively. The use of beta-MePhe as a replacement for Phe3 was based upon the desire to reduce the conformational flexibility of the Phe3 side chain by imposing a steric rotational constraint in the form of the beta-methyl substituent and to thus deduce the residue 3 side chain orientation in the delta receptor-bound conformation from the correlation between delta receptor binding affinities and conformational preferences. Molecular mechanics computations revealed, however, that the conformational constraints imposed by the beta-methyl group in the (2S,3S)-MePhe3 and (2S,3R)-MePhe3 analogs were too modest to allow unequivocal determination of delta receptor-bound residue 3 side chain conformation. However, analysis of the high-affinity (2R,3R)-MePhe3 analog revealed a strong preference for a single side chain conformer (chi 1 approximately 60 degrees). Low-energy conformers of this analog could only be effectively superimposed with low-energy conformers of the parent peptide in which the Phe3 side chain conformation was limited to chi 1 approximately -60 degrees. This observation eliminates the last remaining uncertainty regarding conformational features of the pharmacophore elements in the delta receptor-bound state, allowing the proposal of a complete model.

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)

Substitution of aromatic and nonaromatic amino acids for the Phe3 residue in the delta-selective opioid peptide deltorphin I: effects on binding affinity and selectivity.

Deltorphins I and II (Tyr-D-Ala-Phe-Asp-Val-Val-Gly NH2 and Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH2) display a high degree of delta-opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapeptides based on Tyr-c[D-Cys-Phe-D-Pen] (JOM-13). With the goal of development of structure-activity relationships for deltorphins and comparison with that of the cyclic tetrapeptides, ten analogs of deltorphin I were synthesized in which Phe3 was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3-(1-naphthyl)-L-alanine [1-Nal; Ki(mu) = 767 nM, Ki(delta) = 7.70 nM], 3-(2-naphthyl)-L-alanine [2-Nal; Ki(mu) = 1910 nM, Ki(delta) = 49.2 nM], tryptophan [Ki(mu) = 1250 nM, Ki(delta) = 23.9 nM], and 3-(3-benzothienyl)-L-alanine [Bth; Ki(mu) = 112 nM, Ki(delta) = 3.36 nM] were fairly well tolerated at mu- and delta-receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both mu (Ki = 4710 nM) and delta (Ki = 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced mu binding (Ki = 67.8 nM), leaving delta affinity unaffected (Ki = 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [Ki(mu) = > or = 10,000 nM for each, Ki(delta) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both mu (Ki = 322 and 1240 nM, respectively) and delta (Ki = 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr-c[D-Cys-Phe-D-Pen].

Lipophilicity of opioids determined by a novel micromethod.

The lipophilicity of various mu-selective opioids was determined by measuring their distribution between n-octanol and Tris.HCl buffer, pH 7.4, by a procedure requiring submicromolar concentrations (submilligram amounts) of the compounds. After partitioning at 25 degrees C, the concentrations of opioids in the aqueous phase was quantified by their displacement of bound [3H]Tyr-Ala-Gly-(Me)Phe-Gly-ol (DAMGO) from opioid receptor in brain membranes. The obtained distribution coefficients (log Papp) agreed well with respective values determined previously with other, less sensitive or more cumbersome, methods of quantitation. The procedure is precise and versatile, and offers the routine assessment of lipophilicity as part of the in vitro characterization of opioids frequently available in limited quantities. In principle, the method is applicable to any compound whose binding to its receptor is quantifiable.

Tetrachlorodibenzo-p-dioxin alters rat hypothalamic endorphin and mu opioid receptors.

The present study was undertaken to assess if hypothalamic beta-endorphin (beta E) and/or brain mu opioid receptors are associated with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (50 micrograms/kg)-induced hypophagia and body weight decline in rats. Hypothalamic beta E concentrations were initially increased to 166% of controls on day 1, and then were depressed to 39% and 49% of control values on days 2 and 3, respectively. Brain mu opioid receptor number was increased 60% in TCDD-treated rats at day 3 without a change in the binding affinity. Food-restricted rats did not exhibit changes in hypothalamic beta E concentrations or brain mu opioid receptor number. These results indicate that TCDD causes early perturbations in hypothalamic beta E concentrations and brain mu receptor number, which may contribute to the mechanisms by which TCDD leads to decreased food intake and progressive weight loss.

Chronic ethanol consumption depresses hypothalamic-pituitary-adrenal function in aged rats.

In separate experiments, nine (n = 20) and fifteen (n = 12) month old rats were treated with either 6% ethanol or 12% sucrose (to balance caloric intake) in the drinking water to examine the effect of chronic ethanol consumption on the hypothalamic-pituitary-adrenal axis of aged rats. Rats were maintained on these treatment regimens for thirty days and were killed by decapitation. Blood was collected and plasma concentrations of adrenocorticotropin (ACTH) and corticosterone were determined by radioimmunoassay. Adrenal glands were cleaned, quartered and used to test in vitro responsiveness to ACTH. Anterior pituitary glands from all 15 month old rats and one half of the nine month old rats were collected, frozen and extracted for measurement of tissue ACTH concentration. The remaining anterior pituitary glands from the nine month old rats were challenged with corticotropin releasing hormone (CRH) to test in vitro responsiveness. In nine month old rats, chronic ethanol consumption decreased plasma ACTH and corticosterone (P less than 0.05). Pituitary ACTH concentrations were unchanged in treated nine month old rats, but the amount of pituitary ACTH released in response to CRH was decreased (P less than 0.05) in rats consuming ethanol. In vitro responsiveness of the adrenal gland to ACTH in nine month old rats consuming ethanol was unchanged (P greater than 0.05). Plasma ACTH and corticosterone concentrations were also decreased in 15 month old rats chronically consuming ethanol (P less than 0.05). No differences were noted in responsiveness of the adrenal gland or in the amount of pituitary ACTH due to ethanol consumption in 15 month old rats (P greater than 0.05). The results of these experiments indicate that chronic ethanol consumption decreases hypothalamic-pituitary-adrenal function in aged rats.