Effect of brain lesions on [3H]ohmefentanyl binding site densities in the rat striatum and substantia nigra.

We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of beta-endorphin to the isolated renal basolateral membranes in vitro

Binding of human beta-endorphin (beta-EP) to rat renal basolateral membranes was characterized using [125I]Tyr27-beta-EP ([125I]beta-EP) as a primary ligand. Ten millimolar of ethylenediaminetetra acetic acid (EDTA) completely inhibited the degradation of [125I]beta-EP in the incubation mixture at 4 degrees C, thus making it possible to quantitatively examine the [125I]beta-EP binding. The specific binding of [125I]beta-EP to the basolateral membranes was reversible and saturable, and a nonlinear least-squares regression analysis of a saturation isotherm revealed two different classes of specific binding sites. One class had an apparent dissociation constant (Kd) of 0.68 nM and a lower number of binding sites (33 fmol/mg protein), whereas the other class had a lower affinity (apparent Kd of 210 nM) and a higher number of binding sites (7.3 pmol/mg protein). Inhibition of the [125I]beta-EP binding by naloxone (10 microM) was approximately only 20%, and that by D-Ala2-D-Leu5-enkephalin (10 microM) was null, suggesting the major role of a non-opioid binding component in specific [125I]beta-EP binding to basolateral membranes. Moreover, a 50% inhibition by 10 microM of dynorphin(1-13) suggests that a certain region of the primary structure of beta-EP, excluding at least the NH2-terminal enkephalin sequence, is of particular importance for the [125I]beta-EP binding. These lines of evidence suggest the existence of two different classes of specific binding sites for beta-EP on the renal basolateral membranes, and the high-and low-affinity bindings may be attributed to opioid and non-opioid receptors, respectively, as judged by known characteristics of opioid and non-opioid receptors in other peripheral tissues.     

Synthesis of constrained analogues of cholecystokinin/opioid chimeric peptides

In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide mimetic for Nle-Asp was designed and synthesized. Starting from β-allyl substituted aspartic acids, the terminal double bond was oxidized resulting in spontaneous cyclization to form racemic hemiaminals. Allylation of the hemiaminals afforded 5-allyl substituted proline analogues, which on oxidation, Horner-Emmons olefination, asymmetric hydrogenation, and bicyclization afforded bicyclic dipeptide mimetics for Nle-Asp. Constrained CCK/opioid peptide analogues containing bicyclic dipeptide mimetics for Nle-Gly, Nle-Asp, and homoPhe-Gly were then synthesized and analyzed at both the CCK and opioid receptors.     

Circular dichroism study of the carbohydrate-modified opioid peptides

The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II beta-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) beta-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.     

Influence of Leu5 configuration on the equilibrium constants of complexes of [Leu]-enkephalin with β-cyclodextrin studied by fluorescence spectroscopy, microcalorimetry and 1H NMR spectroscopy

Natural enkephalins and their analogues are very important as potential therapeutic agents (analgetics). In this paper we describe the influence of Leu chirality of cyclic [Leu]enkephalins on the binding constant with β-cyclodextrin and spatial and mutual orientation of guest and host molecules. The formation of complexes is enthalpy driven for both cyclic [Leu]enkephalins. Moreover, d-configuration of Leu residue causes an increase of the binding constant of cyclic enkephalin compared to l-analogue. An analysis of 2D NMR spectra reveals that, apart from inclusion complex formed by penetration of cyclodextrin cavity from wider and narrow rims by Trp or Leu residue, a side and/or bottom association complexes are formed.     

Endomorphin analogues with balanced affinity for bothμ- andδ-opioid receptors

Analogues of endomorphin and tripeptidcs modified at positions 4 and 3,respectively,with various phenylalanine analogues were synthesized and their affinities for opioid receptors were evaluated.Most of the peptides exhibited potentμ-receptor affinity and selectivity,among them,compound 7(Dmt-Pro-Tmp-Tmp-NH_2) exhibited potent affinity for bothμ-andδ-receptors (K_iμ= 0.47 nmol/L,K_iδ= 1.63 nmol/L).     

Determination of enkephalin peptides by nonaqueous capillary electrophoresis with electrochemical detection

Nonaqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the analysis of enkephalin peptides. The effect of different buffer compositions on the electrophoretic behavior of methionine enkephalin, leucine enkephalin, and [D-Ala2]-leucine enkephalin was studied. Separation of the protonated and the deprotonated peptides was obtained using ACN/methanol-based electrolyte systems. The electrochemical behavior of the enkephalins was studied by the capillary batch injection analysis technique. NACE-ED yielded well-defined signals in the oxidation mode only for the negatively charged analytes. The optimized BGE for the counterelectroosmotic separation consisted of 10 mM ammonium acetate in ACN/methanol (3:1 v/v). Using a platinum microdisk electrode set to an actual potential of +0.65 V detection limits in the submicromolar range were observed which are about one order of magnitude lower compared to UV detection. Problems concerning EOF instability and electrode fouling caused by water and other neutral sample impurities transported by the EOF can be avoided in the EOF-inverted mode using poly(ethylene glycol)-coated capillaries and an actual working electrode potential of +1.0 V. For the quantification of the enkephalins [D-Ala2]leucine enkephalin was used as internal standard. The practical utility for the determination of enkephalins in spiked plasma samples after SPE was demonstrated.     

Resonance Energy Transfer Relates the Gas‐Phase Structure and Pharmacological Activity of Opioid Peptides

Enkephalins are efficient pain‐relief drugs that bind to transmembrane opioid receptors. One key structural parameter that governs the pharmacological activity of these opioid peptides and is typically determined from condensed‐phase structures is the distance between the aromatic rings of their Tyr and Phe residues. We use resonance energy transfer, detected by a combination of cold ion spectroscopy and mass spectrometry, to estimate the Tyr–Phe spacing for enkephalins in the gas phase. In contrast to the condensed‐phase structures, these distances appear to differ substantially in enkephalins with different pharmacological efficiencies, suggesting that gas‐phase structures might be a better pharmacophoric metric for ligand peptides.     

Influence of Dab2 and Pro3 configuration of [Leu]-enkephalins on the interactions with β-cyclodextrin studied by fluorescence spectroscopy, microcalorimetry and 1H NMR spectroscopy

Natural enkephalins and their analogues are very important as potential therapeutic agents (analgetics). Herein we describe the influence of Dab and Pro chirality of cyclic [Leu]enkephalins (X¹-c[Dab²-Pro³-βNal(2)⁴-Leu⁵], where X = Tyr or Phe) on the binding constant with β-cyclodextrin and spatial and mutual orientation of guest and host molecules. The formation of complexes is enthalpy driven for all cyclic [Leu]enkephalins studied as well as for Nal and AcNalNH₂. Moreover, change of Dab residue configuration has a greater influence on changes of the binding constant of cyclic enkephalin with β-CD than change of Pro chirality has. Also, the replacement of Tyr¹ residue by Phe¹ substantially changes the peptide chain conformation. An analysis of 2D NMR spectra reveals that, apart from inclusion complex formed by penetration of cyclodextrin cavity from wider and narrow rims by Nal, Tyr or Phe or Leu residue, a side and/or bottom association complexes are formed.     

Cyclopropane-derived peptidomimetics. Design, synthesis, and evaluation of novel enkephalin analogues

It is known that peptide mimics containing trans-substituted cyclopropanes stabilize extended conformations of oligopeptides, and molecular modeling studies now suggest that the corresponding cis-cyclopropane dipeptide isosteres could stabilize a reverse turn. To begin to assess this possibility, a series of cis-substituted cyclopropanes were incorporated as replacements of the Gly(2)-Gly(3) and Phe(4)-Leu(5) dipeptide subunits in Leu-enkephalin (H(2)N-Tyr-Gly-Gly-Phe-Leu-OH), which is believed to bind to opiod receptors in a conformation containing a beta-turn. General methods for the synthesis of the cyclopropane-containing dipeptide isosteres -XaaPsi[COcpCO]Yaa- and -XaaPsi[NHcpNH]Yaa-were developed by a sequence that featured the enantioselective cyclization of allylic diazoacetates catalyzed by the chiral rhodium complexes Rh(2)[(5S)-MEPY](4) and Rh(2)[(5R)-MEPY](4). A useful modification of the Weinreb amidation procedure was applied to the opening of the intermediate lactones with dipeptides, and a novel method for the synthesis of substituted diaminocyclopropanes was also developed. The Leu-enkephalin analogues were tested in a panel of binding and functional assays, and although those derivatives containing cyclopropane replacements of the Gly(2)-Gly(3) exhibited low micromolar affinity for the mu-receptor, analogues containing such replacements for the Phe(4)-Leu(5) subunit did not bind with significant affinity to any of the opioid receptors. These results are discussed.     

Opioid Peptides Determination by Tyrosinase-Modified Oxygen Electrode

Abstract

Opioid peptides morphiceptin (Tyr-Pro-Phe-Pro-NH₂), [D-Ala², D-Leu⁵]-enkephalin (Tyr-D-Ala-Gly-Phe-D-Leu, DADLE) and [D-Thr²]-Leu-enkephalin-Thr (Tyr-D-Thr-Gly-Phe-Leu-Thr, DTLET) containing tyrosine has been studied in the reaction with mushroom tyrosinase immobilized on Clark-type oxygen electrode. A 4–6-times higher response is observed for tyrosine compared to peptides. The detection limit of the tyrosinase-modified electrode for DADLE, morphiceptin and DTLET was 6 μM, 9 μM and 16 μM, respectively.     

Biological properties of opioid peptides replacing Tyr at position 1 by 2,6-dimethyl-Tyr.

To understand the effect of the replacement of Tyr residue at position 1 in opioid peptides by 2,6-dimethyl-Tyr (Dmt) on the biological property, chiral (D or L) Dmt1 analogs of Leu-enkephalin (Enk) and Tyr-D-Arg-Phe-beta Ala-NH2 (YRFB) were synthesized and their enzymatic stabilities, in vitro bioactivities and receptor binding affinities compared with those of parent peptides. [L-Dmt1]Enk (1) exhibited 4-fold higher stability against aminopeptidase-M and possessed dramatically increased activities in guinea pig ilium (GPI) (187-fold) and mouse vas deferens (MVD) (131-fold) assays, and in rat brain receptor binding assays (356-fold at mu receptor and 46-fold at delta receptor) as compared to Enk. [L-Dmt1]YRFB (3) also exhibited increased activities in GPI (46-fold) and MVD (177-fold) assays, and in the binding assays (69-fold at mu receptro and 341-fold at delta receptor) as compared to the parent peptide. [D-Dmt1]Enk (2) and [D-Dmt1]YRFB (4) exhibited activities with diminished or lesser potency than the parent peptide in all assays. These results indicate that there is a tendency for mu affinity to be enhanced more than delta affinity with introduction of L-Dmt into delta ligand peptide (Enk), and for delta affinity to be enhanced more than mu affinity in case of mu ligand peptide (YRFB), resulting in reduced receptor selectivities at the receptors.     

Identification of opioid-binding materials of rat brain

Digitonin-solubilized opioid receptors from rat brain were purified with an affinity resin, AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin (DADLE). Radioreceptor binding assay showed that the purified materials had specific opioid-binding activity of 310 pmol/mg protein on DADLE binding. Analyses by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that the materials were rich in two polypeptides; the major component had a molecular weight of 62000-64000. To establish the materials responsible for binding opiates, the purified materials were cross-linked with 125I-labeled beta-endorphin using bis[2-(succinimidooxycarbonyloxy)-ethyl]sulfone as a cross-linker. The molecular weight of 62000-64000, the major band of the purified materials on SDS-PAGE, agreed closely with that determined by the cross-linking experiment. The results suggest that the purified materials contained opioid-binding materials (opioid receptors).     

Synthesis of pyrazinone ring-containing opioid mimetics and examination of their opioid receptor-binding activity.

Cyclization of dipeptidyl chloromethyl ketones gave 6-(4-aminobutyl)-3-carboxyethyl-5-methyl-2(1H)-pyrazinone, 3-(4-aminobutyl)-6-carboxyethyl-5-methyl-2(1H)-pyrazinone, and 3,6-bis(4-aminobutyl)-5-methyl-2(1H)-pyrazinone, which were inserted into the enkephalin sequence to give opioid mimetics. Thus, it was confirmed that a pyrazinone ring can be easily inserted into a peptide sequence in order to evaluate structural components required for biologically active peptides.     

Novel Delta‐Opioid‐Receptor‐Selective Ligands in the 14‐Alkoxy‐Substituted Indolo‐ and Benzofuromorphinan Series

Several new indolo‐ and benzofuromorphinans substituted at the positions 5 and 14 were prepared and tested in vitro by means of opioid‐receptor binding and functional ([³⁵S]GTPγS binding) assays. All compounds 1 – 11 displayed high affinity for δ opioid‐binding sites (Table 1). Compound 4 proved to be an agonist, and all other compounds were antagonists. The presence of a Me group at position 5 induced no change in δ affinity (see 1 vs. 3 ), but decreased the μ and κ affinities. An EtO group at position 14 conferred a very high affinity and also high selectivity to δ opioid receptors (see 2 and 10 ). Chain elongation of the 14‐alkoxy group resulted in compounds with reduced δ affinity and selectivity (see 4 and 11 and also 5 – 9 ). The results of the present study indicate that the 5‐ and 14‐positions of indolo‐ and benzofuromorphinans represent critical sites that could be a trigger to develop new compounds with increased δ affinity and/or selectivity.     

Evidence for effect of GM1 on opioid peptide conformation: NMR study on leucine enkephalin in ganglioside-containing isotropic phospholipid bicelles

Enkephalins are endogenous neuropeptides that have opioid-like activities and compete with morphines for the receptor binding. The binding of these neuropeptides to membrane appears crucial since enkephalins interact with the nerve cell membranes to achieve bioactive conformations that fit onto multiple receptor sites (micro, delta, and kappa). Using NMR spectroscopy, we have determined the solution structure of the small opiate pentapeptide leucine enkephalin in the presence of isotropic phospholipid bicelles: phosphocholine bicelles (DMPC:CHAPS 1:4) and phosphocholine bicelles doped with ganglioside GM1 (DMPC:CHAPS:GM1 1:4:0.3). Bicelles containing GM1 were found to interact strongly with leucine enkephalin, whereas a somewhat weaker interaction was observed in the case of bicelles without GM1. Structure calculation from torsion angles, chemical shifts, and NOE-based distance constraints explored that the peptide could flexibly switch between several mu- and delta-selective conformations in both the bicelles though micro-selective conformations turned out to be geometrically preferred in each bicellar system. A detailed analysis of the structures presented supports the variance over the singly associated conformation of enkephalin in nerve cell membranes.     

Synthesis of 2′-aminoalkyl-1-benzylisoquinoline derivatives and medium sized ring analogues with mu opiod receptor binding activities

Novel 2′-aminoalkyl-1-benzylisoquinoline compounds and medium size ring analogues have been prepared using reductive alkylation methods. Four of these analogues were tested for biological activity across 48 different CNS receptors and were showed to have binding activities at the mu opiod receptor.     

Solid phase synthesis and opioid receptor binding properties of presumable dermorphin precursor derivatives.

Presumable dermorphin precursor peptide derivatives comprised of 35 amino acids and their fragments, which are based on the amino acid sequence determined by recombinant deoxyribonucleic acid (DNA) techniques, were synthesized by the solid phase method. A 35-residue peptide amide containing L-Ala2-dermorphin sequence at the N-terminus (1) as well as its D-Ala2 isomer (2) and the C-terminal 20-residue peptide amide were found to be unexpectedly stable against aminopeptidase M digestion and in rat brain membrane fractions mixture, suggesting that the C-terminal Glu-rich moiety of 1 and 2 serves to protect from enzymatic breakdown. In the opioid receptor binding assay, 2 showed 40 and 25-fold higher affinities than 1 for mu and delta-receptors, respectively. The N-terminal 15-residue peptide fragment of 2 showed greatly increased affinities for both receptors, being one half of those of dermorphin, whereas that of 1 showed low affinities. Opioid receptor binding properties of these synthetic peptides may be useful in investigation of the processing to dermorphin.     

On-line coupling of size exclusion and capillary zone electrophoresis via a reversed-phase C18 trapping column for the analysis of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved. The degradation of the peptides, caused by endogenous peptidases in the matrix, is sufficiently inhibited with imipramine HCl. The assay has a satisfactory linearity and intraday (9.70-16.3%) precision considering the complexity of this multidimensional separation system. The sensitivity of the method, with a concentration limit of quantification of 2.5 nug/mL, is comparable with other CZE assays for peptides and sufficient for the quantification of peptide drugs in biological matrices.     

On-line coupling of SPE and CE-MS for peptide analysis

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.