morphinans and salutaridine

Sinoacutine is a natural isoquinoline alkaloid isolated from traditional Chinese medicine

Topics: Administration, Intravenous; Animals; Chromatography, High Pressure Liquid; Female; Injections, Intravenous; Male; Morphinans; Protein Binding; Rats; Rats, Sprague-Dawley; Tissue Distribution

Stephania yunnanensis H. S. Lo is widely used as an antipyretic, analgesic and anti-inflammatory herbal medicine in SouthWest China. In this study, we investigated the anti-inflammatory activity and mechanism of sinoacutine (sino), one of the primary components extracted from this plant.. A RAW264.7 cell model was established using lipopolysaccharide (LPS) induced for estimation of cytokines in vitro, qPCR was used to estimate gene expression, western blot analysis was used to estimate protein level and investigate the regulation of NF- κB, JNK and MAPK signal pathway. In addition, an acute lung injury model was established to determine lung index and levels of influencing factors.. Taken together, our results demonstrate that sino is a promising drug to alleviate LPS-induced inflammatory reactions.

Topics: Acute Lung Injury; Animals; Animals, Outbred Strains; Anti-Inflammatory Agents; China; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mice; Molecular Structure; Morphinans; NF-kappa B; Plant Extracts; RAW 264.7 Cells

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Topics: Aldehyde Reductase; Aldo-Keto Reductases; Alkaloids; Base Sequence; Benzylisoquinolines; Bromoviridae; Carbohydrate Epimerases; Cytochrome P-450 Enzyme System; Escherichia coli; Exons; Gene Expression Regulation, Plant; Gene Fusion; Introns; Ligases; Molecular Sequence Data; Morphinans; Morphine; Open Reading Frames; Opium; Oxidation-Reduction; Papaver; Plant Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Stereoisomerism

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.

Topics: Animals; Benzylisoquinolines; Biosynthetic Pathways; Carbohydrate Metabolism; Codeine; Genetic Engineering; Hydrocodone; Morphinans; Papaver; Saccharomyces cerevisiae; Thebaine

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.

Topics: Alkaloids; Isoenzymes; Molecular Sequence Data; Morphinans; Morphine; Papaver; Plant Cells; Plant Proteins; Proteomics; Tetrahydroisoquinolines; Thebaine

Opium poppy (Papaver somniferum) remains the sole commercial source for several pharmaceutical alkaloids including the narcotic analgesics codeine and morphine, and the semi-synthetic drugs oxycodone, buprenorphine and naltrexone. Although most of the biosynthetic genes have been identified, the post-transcriptional regulation of the morphinan alkaloid pathway has not been determined. We have used virus-induced gene silencing (VIGS) as a functional genomics tool to investigate the regulation of morphine biosynthesis via a systematic reduction in enzyme levels responsible for the final six steps in the pathway. Specific gene silencing was confirmed at the transcript level by real-time quantitative PCR (polymerase chain reaction), and at the protein level by immunoblot analysis using antibodies raised against salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridine 7-O-acetyltransferase (SalAT), thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), and codeine O-demethylase (CODM). In some cases, silencing a specific biosynthetic gene resulted in a predictable accumulation of the substrate for the corresponding enzyme. Reduced SalSyn, SalR, T6ODM and CODM protein levels correlated with lower morphine levels and a substantial increase in the accumulation of reticuline, salutaridine, thebaine and codeine, respectively. In contrast, the silencing of genes encoding SalAT and COR resulted in the accumulation of salutaridine and reticuline, respectively, which are not the corresponding enzymatic substrates. The silencing of alkaloid biosynthetic genes using VIGS confirms the physiological function of enzymes previously characterized in vitro, provides insight into the biochemical regulation of morphine biosynthesis, and demonstrates the immense potential for metabolic engineering in opium poppy.

Topics: Agrobacterium tumefaciens; Alcohol Oxidoreductases; Benzylisoquinolines; Chromatography, High Pressure Liquid; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene Silencing; Genes, Plant; Genetic Vectors; Immunoblotting; Metabolic Engineering; Morphinans; Morphine; NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; Oxidoreductases; Papaver; Plant Proteins; Plant Viruses; Reverse Transcriptase Polymerase Chain Reaction; Substrate Specificity

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

Topics: Alcohol Oxidoreductases; Animals; Aryl Hydrocarbon Hydroxylases; Benzylisoquinolines; Codeine; Cytochrome P-450 CYP2D6; Cytochrome P450 Family 2; Humans; Kinetics; Mass Spectrometry; Microsomes, Liver; Morphinans; Morphine; Phenols; Rats; Rats, Wistar; Substrate Specificity; Thebaine

The biosynthesis of morphine, a stereochemically complex alkaloid, has been shown to occur in plants and animals. A search in the human genome for methyltransferases capable of catalyzing the N-methylation of benzylisoquinoline alkaloids, as biosynthetic precursors of morphine, yielded two enzymes, PNMT (EC 2.1.1.28) and NMT (EC 2.1.1.49). Introduction of an N-terminal poly-histidine tag enabled purification of both proteins by immobilized metal affinity chromatography. Recombinant PNMT and NMT were characterized for their catalytic activity towards four benzylisoquinolines: tetrahydropapaveroline (THP), 6-O-methyl-THP, 4'-O-methyl-THP and norreticuline. Human PNMT accepted none of the offered alkaloids and was only active with its established substrate, phenylethanolamine. The second enzyme, human NMT, converted all four benzylisoquinolines, however, with a strict preference for (R)-configured morphine precursors. Determination of kinetic parameters of NMT for the four (R)-configured benzylisoquinoline alkaloids by LC-MS/MS revealed (R)-norreticuline to be the best substrate with an even higher catalytic activity as compared to the previously reported natural substrate tryptamine. In addition, isolation of the morphine precursor salutaridine from urine of mice injected (i.p.) with (R)-THP provides new evidence that the initial steps of morphine biosynthesis in mammals occur stereochemically and sequentially differently than in plants and suggests an involvement of the herein characterized (R)-specific NMT.

Topics: Alkaloids; Animals; Base Sequence; Benzylisoquinolines; DNA Primers; Ethanolamines; Humans; Isoquinolines; Kinetics; Methyltransferases; Mice; Morphinans; Morphine; Phenylethanolamine N-Methyltransferase; Recombinant Proteins; Stereoisomerism; Substrate Specificity

A method which involves the combination of pH-zone-refining counter-current chromatography (pH-zone-refining CCC) and conventional high-speed counter-current chromatography (HSCCC) was established for the preparative separation of alkaloids from the crude extracts of Stephania kwangsiensis. pH-zone-refining CCC was first performed with the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:1:9, v/v), where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 2.0 g of crude extract, 370 mg of sinoacutine and 600 mg of a mixture of three other alkaloids were obtained. Then, the mixture was further separated by conventional HSCCC with the solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), yielding 42 mg of (-)-crebanine, 50 mg of (-)-stephanine and 30 mg of l-romerine from 150 mg mixture of three other alkaloids, respectively. The purities of the four compounds were all over 98% as determined by HPLC, and the chemical structures of the four compounds were confirmed by positive ESI-MS and (1)H NMR data. Results of the present study successfully indicated that this method was efficient for the preparative separation of alkaloids from natural plants.

Topics: Acetates; Aporphines; Chromatography, High Pressure Liquid; Countercurrent Distribution; Hexanes; Hydrogen-Ion Concentration; Methanol; Morphinans; Nuclear Magnetic Resonance, Biomolecular; Plant Extracts; Spectrometry, Mass, Electrospray Ionization; Stephania; Water

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.

Topics: Animals; Female; Mass Spectrometry; Mice; Mice, Inbred C57BL; Molecular Structure; Morphinans; Morphine; Tetrahydropapaveroline; Thebaine

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425-440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (-)-isoboldine, (-)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (-)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b(5) with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice.

Topics: Animals; Benzylisoquinolines; Catalysis; Cell Line, Tumor; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Humans; Iron; Mice; Morphinans; Morphine; Oxidation-Reduction; Phenols; Rats

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.

Topics: Amino Acid Sequence; Animals; Catalysis; Cell Line; Cytochrome P-450 Enzyme System; Gene Expression; Molecular Sequence Data; Morphinans; Morphine; Papaver; Plant Proteins; Recombinant Proteins; Spodoptera

Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel alpha-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.

Topics: Biosynthetic Pathways; Catalysis; Catalytic Domain; Circular Dichroism; Kinetics; Models, Molecular; Molecular Structure; Morphinans; Morphine; Mutagenesis, Site-Directed; Mutation; NADP; Oxidoreductases; Plant Proteins; Protein Binding; Protein Structure, Tertiary; Substrate Specificity

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

Topics: Alkaloids; Amino Acid Sequence; Cluster Analysis; DNA, Complementary; Expressed Sequence Tags; Gene Expression Profiling; Molecular Sequence Data; Morphinans; Morphine; Oxidoreductases; Papaver; Phylogeny; Plant Proteins; Recombinant Fusion Proteins; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Sequence Analysis, Protein; Substrate Specificity

Seven bio-active alkaloids (stepholidine, sinoacutine, isocorydine, l-tetrahydropalmatine, crebanine, fanchinoline and tetrandrine) in Stephania plants were determined by RP-HPLC, using UV detection (282 nm) and gradient elution. The reversed phase system consisted of ODS column and methanol-water-triethylamine as mobile phase. The flow rate was 1.0 ml.min-1. Good linearity between peak heights and concentrations of the alkaloids was obtained in the concentration range. The HPLC method proved accurate, precise and sensitive. The results showed that there were some differences in the occurrence and content of the alkaloids between various species and between the same species from different habitats and collecting time. Based on the results, some species with high content of the 7 bio-active alkaloids were selected. The study provided some useful information for the utilization of medicinal plant resources in the genus Stephania.

Topics: Alkaloids; Aporphines; Benzylisoquinolines; Berberine; Berberine Alkaloids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Morphinans; Species Specificity; Stephania

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.

Topics: Animals; Chemical Phenomena; Chemistry; Circular Dichroism; Methylation; Morphinans; Morphine; Rats; Thebaine

The alkaloid 5,6,8,14-tetradehydro-4-hydroxy-3, 6-dimethoxy-17-methyl-morphinan-7-one[+/-)-salutaridine) was found to possess 3H-gamma-aminobutyric acid (3H-GABA) displacing activity (IC50 less than 1 mumol/l) in rat brain synaptic membranes. The enhancement of specific 3H-diazepam binding by increasing concentration of (+/-)-salutaridine follows a maximum curve indicating (+/-)-salutaridine to be a partial agonist at the GABA/benzodiazepine receptor complex.

Topics: Animals; Bicuculline; Diazepam; gamma-Aminobutyric Acid; In Vitro Techniques; Morphinans; Rats; Receptors, GABA-A; Synaptic Membranes; Synaptosomes

A phytochemical investigation of Glaucium contortuplicatum Boiss. (Papaveraceae) resulted in the isolation of sinoacutine from this plant for the first time. Spectral evidence for the identity of the isolated compound as sinoacutine is presented.

Topics: Morphinans; Plants

Topics: Alkaloids; Chemistry, Pharmaceutical; Morphinans; Pharmacy; Research