Microsomal Preparations Research Articles

Paraoxonase 1 Q192R Variant and Clopidogrel Efficacy

Clopidogrel, in combination with aspirin, is the standard of care for preventing ischemic cardiovascular events in patients with coronary artery disease, especially those who undergo percutaneous coronary intervention (PCI). Despite its widespread use, significant interindividual variability in clopidogrel response is consistently observed, and recent studies have suggested that as much as 70% of this variability can be attributed to genetic factors.1,2 Article see p 250 Clopidogrel is a thienopyridine prodrug that once activated, exerts its antiplatelet effect by irreversibly binding to the P2Y12 receptor on the surface of platelets inhibiting ADP-stimulated platelet aggregation. Clopidogrel activation requires a 2-step conversion by hepatic enzymes, most notably CYP2C19, whereas esterases (eg, carboxylesterase 1 and carboxylesterase 2) lead to the production of biologically inactive carboxylic acid derivatives.3 Common loss-of-function variants in CYP2C19 are associated with lower active metabolite levels, higher residual on-clopidogrel ADP-stimulated platelet aggregation, and poorer cardiovascular outcomes.3 However, this variant explains only ≈12% of the variation in platelet response to clopidogrel, leaving most of the heritable variation unknown. Recently, Bouman and colleagues4 observed through in vitro studies that paraoxonase 1 (PON1) was the major enzyme in converting 2-oxo-clopidogrel to the active thiol metabolite. Moreover, they reported that a common missense variant in PON1 , Q192R, was a major determinant of clopidogrel efficacy, accounting for nearly 70% of the variability in ADP-stimulated platelet aggregation postclopidogrel treatment. 192Q PON1 had lower hydrolysis efficiency for 2-oxo-clopidogrel compared to 192R PON1 in microsomal preparations, and clopidogrel-treated PCI patients homozygous for the 192Q allele had a significantly higher rate of stent thrombosis compared with patients carrying at least 1 copy of the 192R allele. Curiously, no effect of the well-described CYP2C19 *2 loss-of-function variant on clopidogrel response was observed in the same patient sample. In the past year, our group and …

Modulation of Hepatic Drug Metabolizing Enzymes by Dietary Doses of Thymoquinone in Female New Zealand White Rabbits

Herbal medicines can affect drug metabolizing enzymes. Therefore the effect of thymoquinone (TQ), the active ingredient of black seeds, was examined on rabbit liver drug metabolizing enzymes. Two groups of New Zealand female rabbits received TQ at 10 and 20 mg/kg/day orally and a control group of six animals each were killed after 8 weeks. Blood and livers were harvested and the activity of cytochrome P450 (CYP) and phase II enzymes in the microsomal and cytosolic preparations were measured by HPLC and ELISA methods. The liver enzymes ALT/AST and albumin were similar in the three groups. CYP1A2, CYP3A4, but not CYP2E1, were significantly diminished by TQ treatment. Of the phase II enzymes, glutathione-S-transferase (GST) and glutathione peroxidase (GPx) were significantly induced by the high TQ dose, while the total glutathione levels were unaffected. Glutathione reductase (GR), on the other hand, was significantly induced in the two experimental groups. Thymoquinone has differential effects on CYP and phase II enzymes. Inhibition of some CYP enzyme activities may lead to a hazardous herb-drug interaction. Induction of GR activity may explain the salutatory effect of the black seeds in inhibiting the generation of bioactive metabolites known to promote carcinogenesis and oxidative cell damage.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Discovery and evaluation of spirocyclic derivatives as antagonists of the neuropeptide Y5 receptor

A novel series of spirocyclic derivatives was synthesized and evaluated as NPY Y5R antagonists for the treatment of obesity. Cis and trans analogs 7a and 8a were equipotent in a Y5R binding assay (Ki’s⩽1nM) and displayed good stability in human and rat liver microsome preparations. Compound 7a failed to demonstrate weight loss activity in a diet-induced obese (DIO) rat model at unbound drug levels in the brain that exceeded the Y5R Ki value by 25-fold over a 24-h time-period.

In Vitro and In Vivo Metabolism and Pharmacokinetics of BMS-562086, a Potent and Orally Bioavailable Corticotropin-Releasing Factor-1 Receptor Antagonist

The absorption, distribution, metabolism, and excretion (ADME) and the pharmacokinetic characteristics of BMS-562086 [pexacerfont; 8-(6-methoxy-2-methyl-3-pyridinyl)-2,7-dimethyl-N-[(1R)-1-methylpropyl]pyrazolo(1,5-a)-1,3,5-triazin-4-amine (DPC-A69448)] were investigated in vitro and in animals to support its clinical development. BMS-562086 was orally bioavailable in rats, dogs, and chimpanzees, with an absolute oral bioavailability of 40.1, 58.8, and 58.5%, respectively. BMS-562086 was extensively metabolized in hepatocytes from all species and completely metabolized in rats. The primary biotransformation pathways found for BMS-562086 in both liver microsomal and hepatocyte preparations and in rats were similar. These included O-demethylation, hydroxylation at the N-alkyl side chain and N-dealkylation. Multiple cytochromes P450 including CYP3A4/5 were involved in the metabolic clearance of BMS-562086. Both renal and biliary excretion played a significant role in elimination of the metabolites of BMS-562086. The involvement of other metabolic enzymes in addition to CYP3A4/5 in elimination of BMS-562086 suggests a reduced potential for drug-drug interaction through modulation of CYP3A4/5. Chimpanzees proved to be a good animal model in predicting BMS-562086 human clearance. Virtual clinical trials performed with a population-based ADME simulator suggested that a minimal dose of 100 mg daily would provide sufficient drug exposure to achieve plasma concentrations above the projected human efficacious plasma concentration of BMS-562086 (> 500 nM). In summary, BMS-562086 exhibited favorable ADME and pharmacokinetic properties for further development.

Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B

Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

Suggesting a testing strategy for possible endocrine effects of drug metabolites

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an “effect-based” and a “compound-by-compound” testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the “effect-based” approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the “compound-by-compound” testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.

Identification of Human Cytochrome P450 and Flavin-Containing Monooxygenase Enzymes Involved in the Metabolism of Lorcaserin, a Novel Selective Human 5-Hydroxytryptamine 2C Agonist

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze the formation of four primary oxidative metabolites of lorcaserin, suggesting that lorcaserin has a low probability of drug-drug interactions by concomitant medications.

Thermal dependence of cardiac SR Ca2+-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca2+ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca2+ uptake and E–C coupling. Ca2+ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca2+ uptake as low as 5°C. As a consequence, the Q10 values in coho salmon were low over a range of different temperature intervals. Maximal Ca2+ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca2+ uptake activity at 35°C, whereas the fish (tuna and salmon) had maximal activity at 30°C. At 35°C, the rate of Ca2+ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35°C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.

Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2

BackgroundThe Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis.MethodsBiochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity.ResultsThrough microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11.ConclusionsCollectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool.

Comparative Study of the Cytotoxicity and Genotoxicity of Alpha- and Beta-Asarone

The cytotoxicity of alpha- and beta-asarone was investigated with the BrdU assay in HepG2-cells. Alpha-asarone was found to be more toxic than beta-asarone after 24 hours of treatment. Investigation of the genotoxicity using the micronucleus assay in the HepG2-cell system showed that only after metabolic activation by a liver microsomal preparation, beta-asarone was able to induce micronuclei at concentrations higher than 50 μg/mL.

Biotransformation of endosulfan by the tiger worm, <i>Eisenia fetida</i>

This study assesses the role of the earthworm, Eisenia fetida, in the breakdown of endosulfan in a soil environment. Two strains of E. fetida were used in this study to assess the effect of salinity on toxicity and metabolism of endosulfan in these earthworms. One strain of E. fetida (R) was reared in high salinity soil (over 2.0 dS/m of electric conductivity) from Shiwha lake, Korea. A control strain (W) was reared in pig manure compost. Acute toxicity of endosulfan was lower in the R strain when endosulfan was injected. In vitro metabolic studies of endosulfan based on microsomal preparations showed that both strains produced two major metabolites, endosulfan sulfate and endosulfan diol. The production rate of endodulfan sulfate was not significantly different between the strains, while endosulfan diol production was significantly different. In vivo metabolism studies showed only one primary metabolite, endosulfan sulfate, was produced by both strains. HPLC-MS/MS analysis showed annetocin was the indicative protein newly expressed in the R strain in relation to salinity exposure. These findings suggest salinity may induce hydrolyzing enzymes to produce endosulfan diol from endosulfan.

Development of an In Vitro System with Human Liver Microsomes for Phenotyping of CYP2C9 Genetic Polymorphisms with a Mechanism-Based Inactivator

Polymorphisms in cytochrome P450 enzymes can significantly alter the rate of drug metabolism, as well as the extent of drug-drug interactions. Individuals who homozygotically express the CYP2C9*3 allele (I359L) of CYP2C9 exhibit ∼70 to 80% reductions in the oral clearance of drugs metabolized through this pathway; the reduction in clearance is ∼40 to 50% for heterozygotic individuals. Although these polymorphisms result in a decrease in the activity of individual enzyme molecules, we hypothesized that decreasing the total number of active enzyme molecules in an in vitro system (CYP2C9*1/*1 human liver microsomes) by an equivalent percentage could produce the same net change in overall metabolic capacity. To this end, the selective CYP2C9 mechanism-based inactivator tienilic acid was used to reduce irreversibly the total CYP2C9 activity in human liver microsomes. Tienilic acid concentrations were effectively titrated to produce microsomal preparations with 43 and 73% less activity, mimicking the CYP2C9*1/*3 and CYP2C9*3/*3 genotypes, respectively. With probe substrates specific for other major cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), no apparent changes in the rate of metabolism were noted for these enzymes after the addition of tienilic acid, which suggests that this model is selective for CYP2C9. In lieu of using rare human liver microsomes from CYP2C9*1/*3 and CYP2C9*3/*3 individuals, a tienilic acid-created knockdown in human liver microsomes may be an appropriate in vitro model to determine CYP2C9-mediated metabolism of a given substrate, to determine whether other drug-metabolizing enzymes may compensate for reduced CYP2C9 activity, and to predict the extent of genotype-dependent drug-drug interactions.

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

In vitro drug metabolism by C-terminally truncated human flavin-containing monooxygenase 3

Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K m values of 45 ± 8 μM and 25 ± 4 μM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.

2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

The Cooperative Activities of CSLD2, CSLD3, and CSLD5 Are Required for Normal Arabidopsis Development

Glycosyltransferases of the Cellulose Synthase Like D (CSLD) subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/ csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were co-expressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.

AXY8 Encodes an α-Fucosidase, Underscoring the Importance of Apoplastic Metabolism on the Fine Structure of Arabidopsis Cell Wall Polysaccharides

An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.

Induction of morphine-6-glucuronide synthesis by heroin self-administration in the rat

Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronide (M6G), a potent MOR agonist. We have found that rats that had received repeated intraperitoneal injections of heroin exhibit measurable levels of M6G (which is usually undetectable in this species). The goal of the present study was to investigate whether M6G synthesis can be induced by intravenous (i.v.) heroin self-administration (SA). Rats were trained to self-administer either heroin (50μg/kg per infusion) or saline for 20 consecutive 6-h sessions and then challenged with an intraperitoneal challenge of 10mg/kg of heroin. Plasma levels of heroin, morphine, 6-mono-acetyl morphine, M3G, and M6G were quantified 2h after the challenge. In vitro morphine glucuronidation was studied in microsomal preparations obtained from the liver of the same rats. Heroin SA induced the synthesis of M6G, as indicated by detectable plasma levels of M6G (89.7 ± 37.0ng/ml vs. 7.35 ± 7.35ng/ml after saline SA). Most important, the in vitro V (max) for M6G synthesis was correlated with plasma levels of M6G (r (2) = 0.78). Microsomal preparations from saline SA rats produced negligible amounts of M6G. Both in vivo and in vitro data indicate that i.v. heroin SA induces the synthesis of M6G. These data are discussed in the light of previous studies conducted in heroin addicts indicating that in humans heroin enhances the synthesis of the active metabolite of heroin and morphine.