RESUMO
The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.
Assuntos
Codeína/biossíntese , Morfina/biossíntese , Papaver/metabolismo , Hidrocodona/análogos & derivados , Hidrocodona/metabolismo , Isomerases/fisiologia , Ópio/metabolismo , Papaver/enzimologia , Tebaína/metabolismoRESUMO
Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.
Assuntos
Ligases/metabolismo , Papaver/enzimologia , Proteínas de Plantas/metabolismo , Tebaína/metabolismo , Cristalografia por Raios X , Ligases/química , Modelos Moleculares , Papaver/química , Papaver/metabolismo , Proteínas de Plantas/química , Conformação Proteica , Teoria QuânticaRESUMO
The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Tebaína/metabolismo , Conformação Molecular , Proteínas de Saccharomyces cerevisiae/química , Tebaína/químicaRESUMO
The biosynthesis path engineering could be very promising for mass production of alkaloids by applying elicitors in the cell suspension culture of Persian poppy (Papaver bracteatum Lindl.). In this work, the effects of different concentrations of methyl jasmonate (MJ) and phloroglucinol (PG) on thebaine and sanguinarine productions in vitro were investigated. Roots as explant and supplementing 3 mg L-1 2,4-Dichlorophenoxyacetic acid with 0.5 mg L-1 Benzyl amino purine to modified MS medium were selected to achieve the most efficient combination for callus induction and production of callus fresh and dry weights. At 48 h after treatment, the addition of PG and MJ individually and in combination together significantly increased both thebaine and sanguinarine contents than the control. The results of high-performance liquid chromatography (HPLC) detection indicated that the highest production rate has been achieved through a synergic effect of two elicitors after 48 h. Results revealed that adding 200 µM of MJ and 100 mg L-1 PG increased thebaine and sanguinarine contents by 56.36 and 107.71-fold than control cells, respectively.
Assuntos
Acetatos/farmacologia , Benzofenantridinas/biossíntese , Técnicas de Cultura de Células/métodos , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Papaver/metabolismo , Floroglucinol/farmacologia , Tebaína/metabolismo , Biomassa , Cromatografia Líquida de Alta Pressão , Isoquinolinas , Papaver/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , SuspensõesRESUMO
Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O(3)- and the O(6)-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O(3)-demethylation and the O(6)-demethylation are members of the Fe(II)/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O(3)-demethylation. We report that demethylation of thebaine at the O(6)-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O(6)-demethylation of thebaine by an Fe(II)/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O(6)-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O(6)-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.
Assuntos
Citocromo P-450 CYP3A/metabolismo , Morfina/biossíntese , Tebaína/metabolismo , Animais , Biocatálise , Encéfalo/metabolismo , Humanos , Masculino , Metilação , Microssomos Hepáticos/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
Immunofluorescence labeling and shotgun proteomics were used to establish the cell type-specific localization of morphine biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)-reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular localization of morphine biosynthesis in opium poppy.
Assuntos
Morfina/biossíntese , Papaver/citologia , Células Vegetais/enzimologia , Proteínas de Plantas/metabolismo , Alcaloides/metabolismo , Isoenzimas/metabolismo , Dados de Sequência Molecular , Morfinanos/metabolismo , Papaver/metabolismo , Proteômica , Tetra-Hidroisoquinolinas/metabolismo , Tebaína/metabolismoRESUMO
Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.
Assuntos
Proteínas de Bactérias/metabolismo , Codeína/biossíntese , Morfina/biossíntese , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Pseudomonas putida/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Bactérias/genética , Dosagem de Genes , Expressão Gênica , Hidrocodona/análogos & derivados , Hidrocodona/metabolismo , Hidromorfona/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Oxicodona/metabolismo , Papaver/genética , Proteínas de Plantas/genética , Pseudomonas putida/genética , Saccharomyces cerevisiae/genética , Tebaína/metabolismoAssuntos
Biotecnologia/legislação & jurisprudência , Tráfico de Drogas/legislação & jurisprudência , Tráfico de Drogas/prevenção & controle , Drogas Ilícitas/legislação & jurisprudência , Drogas Ilícitas/metabolismo , Morfina/biossíntese , Saccharomyces cerevisiae/metabolismo , Regulamentação Governamental , Heroína/economia , Heroína/metabolismo , Humanos , Drogas Ilícitas/economia , Engenharia Metabólica , Morfina/economia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Biologia Sintética/legislação & jurisprudência , Tebaína/economia , Tebaína/metabolismoRESUMO
It has been firmly established that humans excrete a small but steady amount of the isoquinoline alkaloid morphine in their urine. It is unclear whether it is of dietary or endogenous origin. There is no doubt that a simple isoquinoline alkaloid, tetrahydropapaveroline (THP), is found in human and rodent brain as well as in human urine. This suggests a potential biogenetic relationship between both alkaloids. Unlabeled THP or [1,3,4-D(3)]-THP was injected intraperitoneally into mice and the urine was analyzed. This potential precursor was extensively metabolized (96%). Among the metabolites found was the phenol-coupled product salutaridine, the known morphine precursor in the opium poppy plant. Synthetic [7D]-salutaridinol, the biosynthetic reduction product of salutaridine, injected intraperitoneally into live animals led to the formation of [7D]-thebaine, which was excreted in urine. [N-CD(3)]-thebaine was also administered and yielded [N-CD(3)]-morphine and the congeners [N-CD(3)]-codeine and [N-CD(3)]-oripavine in urine. These results show for the first time that live animals have the biosynthetic capability to convert a normal constituent of rodents, THP, to morphine. Morphine and its precursors are normally not found in tissues or organs, presumably due to metabolic breakdown. Hence, only that portion of the isoquinoline alkaloids excreted in urine unmetabolized can be detected. Analysis of urine by high resolution-mass spectrometry proved to be a powerful method for tracking endogenous morphine and its biosynthetic precursors.
Assuntos
Morfina/biossíntese , Morfina/urina , Animais , Feminino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfina/química , Tetra-Hidropapaverolina/química , Tetra-Hidropapaverolina/metabolismo , Tebaína/metabolismoRESUMO
Systematic screening of morphine pathway intermediates in engineered yeast revealed key biosynthetic enzymes displaying potent feedback inhibition: 3'-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT), which yields (S)-reticuline, and the coupled salutaridinol-7-O-acetyltransferase (SalAT) and thebaine synthase (THS2) enzyme system that produces thebaine. The addition of deuterated reticuline-d1 to a yeast strain able to convert (S)-norcoclaurine to (S)-reticuline showed reduced product accumulation in response to the feeding of all four successive pathway intermediates. Similarly, the addition of deuterated thebaine-d3 to a yeast strain able to convert salutaridine to thebaine showed reduced product accumulation from exogenous salutaridine or salutaridinol. In vitro analysis showed that reticuline is a noncompetitive inhibitor of 4'OMT, whereas thebaine exerts mixed inhibition on SalAT/THS2. In a yeast strain capable of de novo morphine biosynthesis, the addition of reticuline and thebaine resulted in the accumulation of several pathway intermediates. In contrast, morphine had no effect, suggesting that circumventing the interaction of reticuline and thebaine with 4'OMT and SalAT/THS2, respectively, could substantially increase opiate alkaloid titers in engineered yeast.
Assuntos
Morfina , Papaver , Vias Biossintéticas , Retroalimentação , Morfina/metabolismo , Saccharomyces cerevisiae/metabolismo , Tebaína/metabolismoRESUMO
A whole cell Escherichia coli biotransformation platform converting thebaine to oripavine and codeine to morphine was demonstrated with industrially applicable yields (â¼1.2 × 10-2 g L-1 h-1 or â¼1.2 × 10-1 g L-1 h-1), improving >13 400-fold upon morphine production in yeast. Mutations enhanced enzyme performance and the use of a purified substrate with rich raw poppy extract expanded applicability.
Assuntos
Codeína , Morfina , Tebaína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
The search for selective kappa opioid receptor (κOR) agonists with an improved safety profile is an area of interest in opioid research. In this work, a series of m-substituted analogs were designed, synthesized, and assayed, resulting in the identification of compound 6c (SLL-1206) as a κOR agonist with single-digit nanomolar activities. The subtype selectivity of compound 6c appeared to be a consequence of an enormous decrease in the affinity for µOR and δOR, rather than a significant increase in the affinity for κOR, which was not the case for SLL-039, another selective and potent κOR agonist identified in our previous work. Besides reduced central nervous system effects, SLL-1206 exhibited substantially improved physicochemical and pharmacokinetic properties compared with SLL-039, with increases of over 20-fold in aqueous solubility and approximately 40-fold in oral bioavailability in rats.
Assuntos
Analgésicos Opioides/uso terapêutico , Dor/tratamento farmacológico , Receptores Opioides kappa/agonistas , Tebaína/análogos & derivados , Tebaína/uso terapêutico , Analgésicos Opioides/síntese química , Analgésicos Opioides/metabolismo , Animais , Células CHO , Cricetulus , Temperatura Alta , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Ratos Sprague-Dawley , Receptores Opioides kappa/metabolismo , Tebaína/metabolismoRESUMO
The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for 'thebaine oripavine poppy 1'), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the world's licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.
Assuntos
Analgésicos Opioides/metabolismo , Morfina/metabolismo , Mutação/genética , Papaver/genética , Papaver/metabolismo , Tebaína/análogos & derivados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Papaver/química , Fenótipo , Tebaína/metabolismoRESUMO
Exploiting the immunosuppressive, analgesic and highly addictive properties of morphine could increase the success of a bacterial pathogen. Therefore, we performed sequence similarity searches for two morphine biosynthesis demethylases in bacteria. For thebaine 6-O-demethylase and codeine O-demethylase, we found strong alignments to three (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii) of the six ESKAPE pathogens (Enterococcus faecalis, Staphylococcus aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter species) that are commonly associated with drug resistance and nosocomial infections. Expression of the aligned sequence found in P. aeruginosa (NP_252880.1/PA4191) is upregulated in isolates obtained from cystic fibrosis patients. Our findings provide putative mechanistic targets for understanding the role of morphine in pathogenicity.
Assuntos
Acinetobacter baumannii/enzimologia , Infecção Hospitalar/microbiologia , Enterobacter/enzimologia , Klebsiella pneumoniae/enzimologia , Oxirredutases O-Desmetilantes/genética , Pseudomonas aeruginosa/enzimologia , Staphylococcus aureus/enzimologia , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Codeína/metabolismo , Enterobacter/genética , Humanos , Klebsiella pneumoniae/genética , Derivados da Morfina/metabolismo , Alcaloides Opiáceos/administração & dosagem , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Staphylococcus aureus/genética , Tebaína/metabolismoRESUMO
We aimed to understand the effects of water stress on the alkaloid production in various developmental stages of poppy plants and the effect of stress on the alkaloids content in the capsules. Three stages of the life cycle of Papaver somniferum L. were selected in our studies: Rosette, Flowering and Lancing developmental stages. Four types of water conditions were examined: Control, Withdrawal of Water, 50% Water Supply and Inundation. The morphological monitoring, results of Relative Water Content and proline content were used as indicators of stress. The result of the measurements in poppy leaves show that the secondary metabolites dramatically respond to these stress conditions. The constant water supply was beneficial for the accumulation of alkaloids in the capsules.
Assuntos
Papaver/metabolismo , Cromatografia Líquida de Alta Pressão , Codeína/metabolismo , Secas , Morfina/metabolismo , Papaver/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Prolina/metabolismo , Estresse Fisiológico , Tebaína/metabolismo , Água/metabolismoRESUMO
The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.
Assuntos
Padrões de Herança , Papaver/genética , Folhas de Planta/genética , Caules de Planta/genética , Característica Quantitativa Herdável , Sementes/genética , Alelos , Cruzamentos Genéticos , Genótipo , Vigor Híbrido , Ópio/isolamento & purificação , Ópio/metabolismo , Papaver/anatomia & histologia , Papaver/química , Papaver/metabolismo , Papaverina/biossíntese , Papaverina/isolamento & purificação , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/metabolismo , Caules de Planta/anatomia & histologia , Caules de Planta/metabolismo , Sementes/anatomia & histologia , Sementes/química , Sementes/metabolismo , Tebaína/isolamento & purificação , Tebaína/metabolismoRESUMO
Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.
Assuntos
Analgésicos Opioides/metabolismo , Escherichia coli/genética , Fermentação , Organismos Geneticamente Modificados/genética , Papaver/genética , Tebaína/metabolismo , Acetiltransferases/genética , Benzilisoquinolinas/metabolismo , Codeína/biossíntese , Coptis/genética , Escherichia coli/metabolismo , Glicerol/metabolismo , Hidrocodona/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Morfina/biossíntese , Organismos Geneticamente Modificados/metabolismo , Oxirredutases/genética , Oxicodona/metabolismoRESUMO
Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.
Assuntos
Engenharia Genética/métodos , Hidrocodona/metabolismo , Saccharomyces cerevisiae/enzimologia , Tebaína/metabolismo , Animais , Benzilisoquinolinas/metabolismo , Vias Biossintéticas/genética , Metabolismo dos Carboidratos , Codeína/metabolismo , Hidrocodona/química , Morfinanos/química , Morfinanos/metabolismo , Papaver/enzimologia , Papaver/genética , Tebaína/químicaRESUMO
Standard samples of pure (+)-salutaridine and (-)-thebaine required to study the mammalian origin of morphine, were prepared from (+)-6-demethylsalutaridine by published procedures and were characterized by CD spectra and physical data. Reductive N-methylation of (-)-northebaine afforded (-)-thebaine, and when 13C-labeled formalin was used, (-)-thebaine with a 13C label on the N-methyl carbon atom resulted. The latter represents a model procedure to prepare ultimately N-14CH3-labeled (-)-thebaine and 14C-labeled congeners.
Assuntos
Morfinanos/metabolismo , Morfina/metabolismo , Tebaína/metabolismo , Animais , Fenômenos Químicos , Química , Dicroísmo Circular , Metilação , RatosRESUMO
The series of experiments reported in this paper examined the spectrum of subtypes of opioid receptors alkylated in vitro by N-cyclopropylmethyl-7 alpha-methylfumaramido-6,14- endoethenotetrahydronororipavine (NIH10236) and four optical isomers of the methylfumaramidophenethyl derivatives of 3-methylfentanyl. Pretreatment of membranes with NIH10236 resulted in a wash-resistant inhibition of the binding of [3H]6 beta-fluoro-6-desoxyoxymorphone (mu binding sites), the binding of [3H][D-ala2,D-leu5]-enkephalin (both the higher and lower affinity delta binding sites) and was without effect on kappa binding sites labelled with [3H]bremazocine. All four potential alkylating derivatives of 3-methylfentanyl were inactive. Pretreatment of membranes with 1 microM of the reversible ligands, (+)-cis-3-methylfentanyl, but not its enantiomer, inhibited the binding of [3H]6 beta-fluoro-6-desoxyoxymorphone and the binding of [3H][D-ala2,D-leu5]enkephalin to the lower affinity binding sites by over 90%. This phenomenon is termed "pseudo-irreversible inhibition." Incubation of pretreated membranes for 60 min at 37 degrees C, in the presence of 200 mM NaCl and 50 microM GppNHp, only partially reversed the masking of opioid receptors by (+)-cis-3-methylfentanyl. For in vivo experiments, membranes were prepared 18-24 hr after the intracerebroventricular administration of 80 and 50 micrograms of NIH10236. This resulted in decreased labelling of mu binding sites, lower affinity [3H][D-ala2,D-leu5]enkephalin binding sites, as well as kappa binding sites, labelled by [3H]U69,593 and [3H]bremazocine. There was no apparent alteration in the higher affinity [3H][D-ala2,D-leu5]enkephalin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)