RESUMEN

BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.

Asunto(s)

Bencilisoquinolinas , Escherichia coli , Ingeniería Metabólica , Ingeniería Metabólica/métodos , Bencilisoquinolinas/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Vías Biosintéticas , Simulación por Computador , Tetrahidropapaverolina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Ácido 3,4-Dihidroxifenilacético/análogos & derivados

RESUMEN

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

Asunto(s)

Aristolochia , Aristolochia/genética , Aristolochia/química , Tetrahidropapaverolina , Metiltransferasas/genética , Filogenia , Plantas

RESUMEN

Morphine can be synthesized endogenously by mammals from dopamine via the intermediate norlaudanosoline. Previously, both compounds have been detected separately in whole brains of mice and brain regions of rats, and in urine of humans. Here, we report a novel method for the analysis of both compounds in single murine brain regions. Initially, a variant of dispersive liquid-liquid microextraction was established by using methanol as an extractant, cyclohexane as solvent, and tributylphosphate as disperser. The extraction method was applied to murine brain regions homogenized with perchloric acid while the subsequent detection was carried out by HPLC with electrochemical detection. In the thalamus of C57Bl/6J mice (n = 3, male, age 4-8 months), morphine and norlaudanosoline could be detected at levels of 19 ± 3.9 and 7.2 ± 2.3 pg/mg, respectively. Overall, we provide a novel method for the simultaneous extraction and detection of both morphine and norlaudanosoline in single murine brain regions.

Asunto(s)

Química Encefálica , Técnicas Electroquímicas/métodos , Microextracción en Fase Líquida/métodos , Morfina/análisis , Tetrahidropapaverolina/análisis , Animales , Encéfalo/metabolismo , Química Encefálica/fisiología , Cromatografía Liquida/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Morfina/metabolismo , Tetrahidropapaverolina/metabolismo

RESUMEN

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

Asunto(s)

Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Escherichia coli/metabolismo , Proteínas de Insectos/metabolismo , Ingeniería Metabólica/métodos , Tetrahidropapaverolina/metabolismo , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Ácido 3,4-Dihidroxifenilacético/metabolismo , Secuencias de Aminoácidos/genética , Animales , Descarboxilasas de Aminoácido-L-Aromático/química , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/aislamiento & purificación , Bombyx , Dopamina/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad

RESUMEN

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.

Asunto(s)

Bencilisoquinolinas/química , Reactores Biológicos/microbiología , Escherichia coli/metabolismo , Laboratorios , Bencilisoquinolinas/metabolismo , Dopamina/metabolismo , Tetrahidropapaverolina/metabolismo

RESUMEN

Noscapine is a potential anticancer drug isolated from the opium poppy Papaver somniferum, and genes encoding enzymes responsible for the synthesis of noscapine have been recently discovered to be clustered on the genome of P. somniferum. Here, we reconstitute the noscapine gene cluster in Saccharomyces cerevisiae to achieve the microbial production of noscapine and related pathway intermediates, complementing and extending previous in planta and in vitro investigations. Our work provides structural validation of the secoberberine intermediates and the description of the narcotoline-4'-O-methyltransferase, suggesting this activity is catalysed by a unique heterodimer. We also reconstitute a 14-step biosynthetic pathway of noscapine from the simple alkaloid norlaudanosoline by engineering a yeast strain expressing 16 heterologous plant enzymes, achieving reconstitution of a complex plant pathway in a microbial host. Other engineered yeasts produce previously inaccessible pathway intermediates and a novel derivative, thereby advancing protoberberine and noscapine related drug discovery.

Asunto(s)

Antineoplásicos/metabolismo , Bioingeniería/métodos , Vías Biosintéticas , Noscapina/metabolismo , Papaver/genética , Saccharomyces cerevisiae , Alcaloides de Berberina , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Descubrimiento de Drogas/métodos , Metiltransferasas/metabolismo , Familia de Multigenes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tetrahidropapaverolina/metabolismo

RESUMEN

The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

Asunto(s)

Bencilisoquinolinas/metabolismo , Alcaloides de Berberina , Enzimas/metabolismo , Biblioteca de Genes , Saccharomyces cerevisiae/metabolismo , Benzofenantridinas/metabolismo , Alcaloides de Berberina/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/biosíntesis , Enzimas/genética , Isoquinolinas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Filogenia , Saccharomyces cerevisiae/genética , Tetrahidropapaverolina/metabolismo , Transcriptoma

RESUMEN

Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

Asunto(s)

Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Hipercinesia/inducido químicamente , Hipercinesia/prevención & control , Tirosina 3-Monooxigenasa/antagonistas & inhibidores , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Actividad Motora/efectos de los fármacos , Neurotransmisores/metabolismo , Tetrahidropapaverolina/farmacología , Pez Cebra

RESUMEN

BACKGROUND: Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine. RESULTS: This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale. CONCLUSIONS: By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.

Asunto(s)

Alcaloides de Berberina/metabolismo , Berberina/análogos & derivados , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/genética , Técnicas de Cultivo Celular por Lotes , Berberina/química , Berberina/metabolismo , Alcaloides de Berberina/química , Reactores Biológicos , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Fermentación , Dosificación de Gen , Proyectos Piloto , Saccharomyces cerevisiae/metabolismo , Tetrahidropapaverolina/química , Tetrahidropapaverolina/metabolismo

RESUMEN

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4'-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6' coupling and four O-methyl groups at C6, C7, C3', and C4' as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3' and 4' hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4'OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4'-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3'-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3'- and 7'-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.

Asunto(s)

Aporfinas/metabolismo , Metiltransferasas/metabolismo , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Bencilisoquinolinas/metabolismo , Alcaloides de Berberina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Isoquinolinas/metabolismo , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Papaveraceae/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/metabolismo , Plantas Medicinales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tetrahidropapaverolina/metabolismo

RESUMEN

Tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) found in diverse pharmaceutical compounds, is used as a starting material for the production of BIA. THP also has various neurobiological properties but is difficult to synthesize. Therefore, a simple method for THP production is desired. Recent studies have shown that microbes, especially bacteria, can serve as platforms for synthesizing these complex compounds; however, because bacteria lack organelles, the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently inhibited or disrupted by undesirable reactions. Indeed, in the first attempt to synthesize THP using a single strain of engineered Escherichia coli, the yield was quite low (<5 µM), mainly because of the oxidation of THP by tyrosinase, an essential enzyme in our production system. To circumvent these problems, we constructed a stepwise (R,S)-THP production system, in which the dopamine-producing step and the subsequent THP-producing step were separated. The yield of (R,S)-THP reached 1.0 mM (287 mg/L), the highest yielding BIA production method using a microbe reported to date. Furthermore, we demonstrated that (R,S)-THP produced by stepwise fermentation is useful for the production of reticuline, an important BIAs intermediate. Based on these observations, applying the stepwise fermentation method is discussed.

Asunto(s)

Fermentación , Ingeniería Metabólica , Monofenol Monooxigenasa/genética , Tetrahidropapaverolina/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Monofenol Monooxigenasa/metabolismo , Tetrahidropapaverolina/metabolismo

RESUMEN

Benzylisoquinoline alkaloids (BIAs) represent a large class of plant secondary metabolites, including pharmaceuticals such as morphine, codeine and their derivatives. Large-scale production of BIA-based pharmaceuticals is limited to extraction and derivatization of alkaloids that accumulate in planta. Synthesis of BIAs in microbial hosts could bypass such limitations and transform both industrial production of BIAs with recognized value and research into uncharacterized BIAs. Here we reconstitute a 10-gene plant pathway in Saccharomyces cerevisiae that allows for the production of dihydrosanguinarine and its oxidized derivative sanguinarine from (R,S)-norlaudanosoline. Synthesis of dihydrosanguinarine also yields the side-products N-methylscoulerine and N-methylcheilanthifoline, the latter of which has not been detected in plants. This work represents the longest reconstituted alkaloid pathway ever assembled in yeast and demonstrates the feasibility of the production of high-value alkaloids in microbial systems.

Asunto(s)

Benzofenantridinas/biosíntesis , Genes de Plantas , Papaver/genética , Saccharomyces cerevisiae/genética , Tetrahidropapaverolina/metabolismo , Transformación Genética/genética , Alcaloides/biosíntesis , Vectores Genéticos , Isoquinolinas , Papaver/metabolismo , Plásmidos , Saccharomyces cerevisiae/metabolismo

RESUMEN

Tetrahydropapaveroline (THP), which is an endogenous neurotoxin, has been suspected to be associated with dopaminergic neurotoxicity of l-DOPA. In this study, we examined oxidative modification of neurofilament-L (NF-L) and neuronal cell death induced by THP. When disassembled NF-L was incubated with THP, protein aggregation was increased in a time- and THP dose-dependent manner. The formation of carbonyl compounds and dityrosine were observed in the THP-mediated NF-L aggregates. Radical scavengers reduced THP-mediated NF-L modification. These results suggest that the modification of NF-L by THP may be due to oxidative damage resulting from the generation of reactive oxygen species (ROS). When THP exposed NF-L was subjected to amino acid analysis, glutamate, proline and lysine residues were found to be particularly sensitive. We also investigated the effects of copper ions on THP-mediated NF-L modification. At a low concentration of THP, copper ions enhanced the modification of NF-L. Treatment of C6 astrocyte cells with THP led to a concentration-dependent reduction in cell viability. When these cells were treated with 100µM THP, the levels of ROS increased 3.5-fold compared with control cells. Furthermore, treatment of cells with THP increased NF-L aggregate formation, suggesting the involvement of NF-L modification in THP-induced cell damage.

Asunto(s)

Muerte Celular , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Tetrahidropapaverolina/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Catecoles/química , Catecoles/aislamiento & purificación , Catecoles/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cobre/química , Cobre/farmacología , Cobre/toxicidad , Depuradores de Radicales Libres/farmacología , Ratones , Peso Molecular , Enfermedades Neurodegenerativas/metabolismo , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/aislamiento & purificación , Neuronas/citología , Neuronas/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tetrahidropapaverolina/química

RESUMEN

Clinical observations stemming from widespread employment of restorative L-3,4-dihydroxyphenylalanine (L-DOPA) therapy for management of dyskinesia in Parkinson's Disease (PD) patients implicate a regulatory role for endogenous morphine in central nervous system dopamine neurotransmission. Reciprocally, it appears that restorative L-DOPA administration has provided us with a compelling in vivo pharmacological model for targeting peripheral sites involved in endogenous morphine expression in human subjects. The biological activities underlying endogenous morphine expression and its interaction with its major precursor dopamine strongly suggest that endogenous morphine systems are reciprocally dysregulated in PD. These critical issues are examined from historical and current perspectives within our short review.

Asunto(s)

Levodopa/uso terapéutico , Morfina/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Animales , Codeína/orina , Humanos , Morfina/biosíntesis , Morfina/orina , Enfermedad de Parkinson/orina , Tetrahidropapaverolina/orina

RESUMEN

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.

Asunto(s)

Morfina/biosíntesis , Morfina/orina , Animales , Femenino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfina/química , Tetrahidropapaverolina/química , Tetrahidropapaverolina/metabolismo , Tebaína/metabolismo

RESUMEN

Tetrahydropapaveroline (THP), a neurotoxic tetrahydroisoquinoline alkaloid formed by condensation between dopamine and dopaldehyde, has been speculated to cause Parkinson's disease and also to contribute to alcohol dependence. Having two catechol moieties, THP may readily undergo oxidation to form an o-quinone intermediate with concomitant production of reactive oxygen species, which can cause neuronal cell death and DNA damage. This review will deal with the current knowledge of neurotoxic effects of this endogenous alkaloid and underlying biochemical mechanisms.

Asunto(s)

Alcoholismo , Catecoles , Muerte Celular , Daño del ADN , Dopamina , Neuronas , Enfermedad de Parkinson , Especies Reactivas de Oxígeno , Tetrahidroisoquinolinas , Tetrahidropapaverolina

RESUMEN

Tetrahydropapaveroline (THP), a dopaminergic isoquinoline neurotoxin, has been reported to contribute to neurodegeneration in parkinsonism. As THP bears two catechol moieties, it undergoes autooxidation or enzymatic oxidation to produce reactive oxygen species (ROS), which may contribute to the THP-induced cell death. Although ROS are cytotoxic, the initial accumulation of ROS may provoke a survival response. In this study, treatment of PC12 cells with THP increased expression of heme oxygenase-1 (HO-1) as an adaptive survival response. Furthermore, THP-induced cytotoxicity was attenuated by the HO-1 inducer (SnCl2) and exacerbated by the HO-1 inhibitor (ZnPP). To elucidate the molecular mechanisms underlying THP-mediated HO-1 expression, we examined the possible involvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regulation of detoxifying/antioxidant genes. THP treatment elevated nuclear translocation of Nrf2 and subsequent binding to antioxidant response element (ARE). PC12 cells transfected with dominant-negative Nrf2 exhibited increased cytotoxicity and decreased HO-1 expression after THP treatment. Moreover, U0126 and LY294002, which are pharmacologic inhibitors of extracellular signal-regulated kinase1/2 and phosphoinositide 3-kinase, respectively, attenuated HO-1 expression as well as Nrf2-ARE binding activity. Taken together, these findings suggest that HO-1 induction via Nrf2 activation may confer a cellular adaptive response against THP-mediated cell death.

Asunto(s)

Adaptación Fisiológica/efectos de los fármacos , Apoptosis/efectos de los fármacos , Hemo-Oxigenasa 1/biosíntesis , Factor 2 Relacionado con NF-E2/metabolismo , Tetrahidropapaverolina/farmacología , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inducción Enzimática , Formazáns/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Potenciales de la Membrana , Modelos Biológicos , Mutación , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Células PC12 , ARN Interferente Pequeño/metabolismo , Ratas , Tetrahidropapaverolina/toxicidad , Sales de Tetrazolio/metabolismo , Factores de Tiempo

RESUMEN

Tetrahydropapaveroline (THP) is a compound derived from dopamine monoamine oxidase-mediated metabolism, particularly present in the brain of parkinsonian patients receiving L-dopa therapy, and is capable of causing dopaminergic neurodegeneration. The aim of this work was to evaluate the potential of THP to cause oxidative stress on mitochondrial preparations and to gain insight into the molecular mechanisms responsible for its neurotoxicity. Our data show that THP autoxidation occurs with a continuous generation of hydroxyl radicals (*OH) and without the involvement of the Fenton reaction. The presence of ascorbate enhances this process by establishing a redox cycle, which regenerates THP from its quinolic forms. It has been shown that the production of *OH is not affected by the presence of either ferrous or ferric iron. Although THP does not affect lipid peroxidation, it is capable of reducing the high levels of thiobarbituric acid-reactive substances obtained in the presence of ascorbate and/or iron. However, THP autoxidation in the presence of ascorbate causes both an increase in protein carbonyl content and a reduction in protein-free thiol content. THP also increases protein carbonyl content when the autoxidation occurs in the presence of iron. The remarkable role played by ascorbate in the production of oxidative stress by THP autoxidation is of particular interest.

Asunto(s)

Encéfalo/metabolismo , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , Tetrahidropapaverolina/metabolismo , Animales , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Carbonilación Proteica , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Tetrahidropapaverolina/farmacología

RESUMEN

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H(2)O(2))-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to (32)P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical ((.)OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to (.)OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5'-TG-3' sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H(2)O(2) and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H(2)O(2) and metals plays an important role in cytotoxicity of L-DOPA.

Asunto(s)

Apoptosis/fisiología , Daño del ADN , Dopamina/metabolismo , Tetrahidropapaverolina/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Bloqueadores de los Canales de Calcio/metabolismo , Bovinos , Cobre/metabolismo , Fragmentación del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Ácido Edético/metabolismo , Compuestos Férricos/metabolismo , Depuradores de Radicales Libres/metabolismo , Células HL-60 , Humanos , Iones , Quelantes del Hierro/metabolismo , Metales/química , Estructura Molecular , Fenantrolinas/metabolismo , Piperidinas/metabolismo

RESUMEN

In this study, 18 polymeric single amino acid and dipeptide surfactants are examined, and their performances, in terms of enantioselectivity, are compared for norlaudanosoline, laudanosoline, laudanosine, chlorthalidone, benzoin, benzoin methyl, and benzoin ethyl enantiomers. Several aspects of amino acid-based polymeric surfactants including comparison of single amino acid versus dipeptide, amino acid order, steric effect, and effect of the position of the chiral center of dipeptide surfactants on the chiral selectivity of these optically active compounds are discussed.

Asunto(s)

Aminoácidos/química , Benzoína/aislamiento & purificación , Clortalidona/aislamiento & purificación , Isoquinolinas/aislamiento & purificación , Micelas , Tetrahidropapaverolina/aislamiento & purificación , Benzoína/química , Estereoisomerismo