Photolabile ligands for opiate receptors

The 2-nitro-4-azidophenyl(NAP)-D-Ala2-Leu5-Enkephalin derivatives: Try-D-Ala-Gly-Phe-Leu CONCH2CH2NH-NAP (E-NAP-EDA) and Try-D-Ala-Gly-Phe-Leu CONCH2CH2NH-COCH2CH2NHNAP(E-NAP- -Ala-EDA) were synthesized by conventional peptide methods. Their structure was determined by amino acid analysis, ultra violet, visible and infra red spectroscopy. Both peptides were shown a) to bind with high affinity to the opiate receptors of rat brain membranes and b) to inhibit strongly the contractions of electrically stimulated vas deferens and the adenyl cyclase of the NG 108-15 cell membranes. These effects were reversed by the antagonist naloxone. Photoloysis of the rat brain membranes-(E-NAP- -Ala-EDA) complex caused a 20-30% inactivation of the opiate receptors. Inactivation was prevented when the complex was irradiated in the presence of naloxone. The radio-labeled derivatives of these enkephalin analogs may prove useful photochemical labels of the opiate receptor.     

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

Method for estimating a compound’s opiate activity based on a versatile three-dimensional model of nonselective opiate pharmacophore

A versatile three-dimensional (3D) model for a nonselective opiate pharmacophore was constructed using an alternative strategy for searching the structural similarity of molecules. The method for calculating the qualitative evaluation of agonistic and antagonistic properties of opiate receptor ligands was developed on this basis. Examples of using this method for evaluating the opiate activity of compounds which essentially differ in their structure from opiates and the traditional opioids are given.     

A protein-lipid model of the opiate receptor

The complexity of the opiate analgesia receptor is indicated by several lines of evidence which are reviewed in this paper: (1) heterogeneity of opiate-receptor interactions; (2) the effect of various enzymatic and chemical treatments of brain membranes on opiate binding; and (3) physico-chemical properties of detergent-extracted membrane components. Based on these findings, a model of the opiate-receptor is proposed which consists of both protein and lipid; the former contains a binding site for enkephalins, and the latter a site for alkaloids, with β-endorphin interacting with both sites. Some implications of this model are briefly discussed.     

Characterization of a plasma factor having opiate and immunoactivity like beta-endorphin.

A factor from human plasma having opiate-like activity was characterized in the present study. In addition to its ability to inhibit the binding of [³H]-methionine-enkephalin to opiate receptors, it also cross-reacted with two β-endorphin specific anti-sera. Compared with β-endorphin, the plasma factor had a shorter action on inhibiting the contraction of the guinea pig ileum. By gel-filtration chromatography, the size of this factor was intermediate between that of β-endorphin and methionine-enkephalin.     

Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

Monoclonal antibody to enkephalins with binding characteristics similar to opiate receptor

Monoclonal antibodies to enkephalins were established by immunization of mice with met-enkephalin, leu-enkephalin or both. Twenty-three clones with a high titer were classified into 6 types according to the binding properties to enkephalins and their derivatives. Antibody LM 239 showed binding characteristics similar to opiate receptor. It has a very high affinity to enkephalins and their derivatives which have a potent opioid activity, but a low affinity to enkephalin derivatives which devoid of opioid activity. The binding of 3H-met-enkephalin to the antibody was inhibited by naloxone and morphine, although the ID50 values were considerably higher than the Ka values of the alkaloids to opiate receptor.     

Capillary gas chromatography of pyrimidines and purines: N,O-peralkyl and trifluoroacetyl-N,O-alkyl derivatives

Preparation and capillary gas chromatographic properties of volatile derivatives of eighteen pyrimidine and purine nucleic acid bases are described. N,O-peralkylation using methylsulfinyl carbanion, methyl or ethyl iodide reagent, and alkylation preceded by N-trifluoroacetylation produced derivatives having minimal adsorption and tailing compared with trimethylsilyl derivatives. Relative retention times and linearity of flame ionization or nitrogen—phosphorus detector response were measured using polar (Superox-FA) and apolar (SE-30) liquid phases. Application of gas chromatography—mass spectrometry to derivatives of DNA hydrolysates using mass chromatography is demonstrated.     

N4-amino-acid derivatives of 6-azacytidine: structure-activity relationship

Several N4-derivatives of 6-azacytidine were synthesized using of Vorbrüggen's condensation method. Their antiviral activity with respect to the adenovirus serotypes 2 and 5 in Hep-2 cells culture was studied and primary specific activity was determined. Correlation between chemical structure of new 6-azacytidine derivatives and their biological properties is discussed.     

Enkephalin-like material in normal human CSF: measurement and levels.

An opioid material in normal human lumbar CSF has been partially characterized and quantified. This material resembles methionine-enkephalin in its behavior in two chromatographic systems. It interacts with the opiate receptor assay and with a methionine-enkephalin radioimmunoassay. Mean human values are 3.12–3.25 pmoles of methionine-enkaphalin equivalents but the median is 1.7–1.9 pmoles. While this material resembles enkephalin, it cannot be identified as such with our current techniques.     

On the specificity of naloxone as an opiate antagonist.

Since the discovery of endogenous opioid peptides in brain (68,69,97,113, 128) and the pituitary gland (26,81,105,125) there has been considerable interest in their possible roles in a variety of physiological and pharmacological processes. Many studies have used antagonism by naloxone as a criterion for implicating endogenous opiates in a process, assuming that naloxene has no pharmacological actions other than those related to blockade of opiate receptors. The doses of naloxene used are often higher than those required to antagonize the analgesic and other effects of morphine. However, multiple forms of opiate receptors are present in nervous tissue and higher concentrations of naloxene are required to antagonize effects mediated by some of these receptors (83). Although the earlier literature supports the assumption that the effects of naloxene are due to the blockade of opiate receptors (87), there are an increasing number of reports which indicate that naloxene may have pharmacological actions unrelated to opiate receptor blockade. The subsequent review serves to emphasize that antagonism by naloxene is a necessary but not sufficient criterion for invoking the mediation of a response by an endogenous opiate (61). Additional lines of evidence which serve to strengthen the conclusion that endogenous opiates mediate a process will be considered.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Selective presence of opiate receptors on intestinal circular muscle cells

Smooth muscle cells isolated separately from the longitudinal and circular muscle layers of guinea pig and human intestine exhibited a unique pattern of response to derivatives of proenkephalin and prodynorphin present in the myenteric plexus. Receptors for other myenteric transmitters (acetylcholine, the octapeptide of cholecystokinin and substance P) capable of mediating contraction, were present on both circular and longitudinal muscle cells, whereas opiate receptors were present on circular muscle cells and selectively absent from longitudinal muscle cells in both species. The opiate myoreceptors belonged to the three main subclasses (kappa, delta and mu) and exhibited a rank order of sensitivity similar to that of opiate neuroreceptors. The distribution of these receptors parallels the distribution of opioid nerve fibers and appears to reflect the role of opioids in the regulation of neuromuscular activity.     

Multiple opiate receptors on neurons of the mammalian central nervous system. In vivo and in vitro studies

Experiments were performed in rat spinal cord cells in vivo and on hippocampal pyramidal cells in vitro. These investigations suggest that acute and chronic treatment renders the neurons subsensitive to opiate alkaloids without altering their sensitivity to opioid peptides. The experiments performed in the dorsal horn of the spinal cord provide evidence that in this structure mu- and delta-receptors may also be localized on the same cell. The evidence for the existence of distinct types of opiate receptors as originally proposed by (1) and suggested by the differing pattern of opiate and opioid peptide activity in various assay systems has been substantiated by investigations involving the selective development of tolerance and the protection of a particular receptor subtype by chemical manipulation. Furthermore, they have been characterized by the use of low concentrations of radiolabelled agonists and antagonists and through the ability of GTP to influence differentially their binding to the opiate receptor (for refs. see: 2). Recently autoradiographic techniques were able to provide direct evidence by mu- and delta-receptors in the mammalian brain (3; 4; 5; 6; and cits. therein). The presence of multiple opiate receptors located on the same cell is suggested by the present study.     

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain.

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.     

Neonatal animal models of opiate withdrawal

The symptoms of opiate withdrawal in infants are defined as neonatal abstinence syndrome (NAS). NAS is a significant cause of morbidity in term and preterm infants. Factors, such as polysubstance abuse, inadequate prenatal care, nutritional deprivation, and the biology of the developing central nervous system contribute to the challenge of evaluating and treating opiate-induced alterations in the newborn. Although research on the effects of opiates in neonatal animal models is limited, the data from adult animal models have greatly contributed to understanding and treating opiate tolerance, addiction, and withdrawal in adult humans. Yet the limited neonatal data that are available indicate that the mechanisms involved in these processes in the newborn differ from those in adult animals, and that neonatal models of opiate withdrawal are needed to understand and develop effective treatment regimens for NAS. In this review, the behavioral and neurochemical evidence from the literature is presented and suggests that mechanisms responsible for opiate tolerance, dependence, and withdrawal differ between adult and neonatal models. Also reviewed are studies that have used neonatal rodent models, the authors' preliminary data based on the use of neonatal rat and mouse models of opiate withdrawal, and other neonatal models that have been proposed for the study of neonatal opiate withdrawal.     

Synthesis of DNA oligodeoxynucleotides containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-adenine adducts of 3,4-epoxy-1-butene

3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD), an important environmental and industrial chemical classified as a probable human carcinogen. The ability of EB to induce point mutations at GC and AT base pairs has been attributed to its reactions with DNA to form covalent nucleobase adducts. Guanine alkylation is preferred at the endocyclic N7 nitrogen, while adenine can be modified at the N1-, N3-, N7-, and the N6 positions. For each of these sites, a pair of regioisomeric 2-hydroxy-3-buten-1-yl and 1-hydroxy-3-buten-2-yl adducts is produced as a result of epoxide ring opening at the terminal C-4 or the internal C-3 carbon position of EB, respectively. The N6-EB-adenine adducts are of particular interest because of their stability in DNA, potentially leading to their accumulation in vivo. In the present work, synthetic DNA oligomers containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-dA (N6-HB-dA) adducts were prepared for the first time by a postoligomerization approach that involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3-buten-2-ol. N6-HB-dA-containing DNA oligomers were isolated by reversed phase HPLC, and the presence of N6-HB-dA in their structure was confirmed by molecular weight determination from HPLC-ESI- -MS of the intact strands and by HPLC-ESI+-MS/MS and MS/MS/MS analyses of the enzymatic digests using synthetic N6-HB-dA as an authentic standard. N6-HB-dA-containing oligomers generated in this study will be used for structural and biological studies.     

Synthesis and antibacterial activity of N4-mono alkyl derivatives of novel glycopeptide LYV07ww01

Thirty-one N(4)-mono alkyl derivatives of novel glycopeptide LYV07ww01 were synthesized by the reductive alkylation and their in vitro antibacterial activity was tested. The benzyl derivatives showed potent activity, especially against vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumoniae.