N-demethylation Activity Research Articles

Chiral resolution of N-methyl-d,l-aspartic acid using a predicted N-demethylase from Chloroflexi bacterium 54-19

N-methyl-d-aspartic acid (NMDA), an amino acid existing in human and animal central nervous system, exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes, Parkinson’s and Alzheimer’s syndrome. In this study, an enzymatic protocol for the chiral resolution of N-methyl-d,l-aspartic acid was built with a predicted N-demethylase (GenBank ID: OJV90073.1) from the genome of Chloroflexi bacterium 54-19. Through sequence alignment, the enzyme shares an identity of 32.19% to 2UZZ (PDB ID) with a conserved catalytic center. Recombinantly expressed in Bacillus subtilis WB600, the N-demethylase was characterized with optimal temperature and pH at 55 ℃ and 7.5, and adaptive temperature and pH were 40–60 ℃ and 6–8. The effects of organic solvents and metal ions were investigated as well. Compared with other previously reported sarcosine oxidases, the enzyme showed a specific N-demethylation activity against N-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography, LC–MS/MS and a detection by polarimeter. The results demonstrated 76% of 4.5 mM N-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L−1 enzyme after reaction of 80 min. This work provided a foundation for mild synthesis of NMDA in industry.Graphical abstract

Promote the expression and corrected folding of an extremely stable N-demethylase by promoter reconstruction, native environment simulation and surface design

Thermomicrobium roseum sarcosine oxidase (TrSOX) was a N-demethylase with specific substrate chiral selectivity, outstanding thermostability and environmental resistance. To promote the expression of TrSOX in Bacillus subtilis W600, the HpaII promoter of pMA5 plasmid was replaced by constitutive or inducible promoters. Through orthogonal experiment, the expression process was optimized, B. subtilis W600 cells containing pMA5-Pxyl-trSOX plasmid were cultivated until OD600nm reached 2.0 and were then induced with 1.6% xylose at 37 °C for 2 h, and the native environment of T. roseum was simulated by heating at 80 °C, with the productivity of TrSOX increased from ~8.3 to ~66.7 μg/g wet cells; and the simulated high temperature was the key switch for the final folding. To reduce the surface hydrophobicity, a S320R mutant was built to form a hydrophilic lid around the entrance of the substrate pocket, and the yield of TrSOX (S320R) was ~163.0 μg/g wet cells, approximately 20 folds as that in the initial expression system. This mutant revealed the similar secondary structure, stability, resistance, chiral substrate selectivity and optimal reaction environment with wild type TrSOX; however, the N-demethylation activities for amino acid derivative substrates were dramatically increased, while those for hydrophobic non-amino acid compounds were repressed.

Soluble expression of Thermomicrobium roseum sarcosine oxidase and characterization of N-demethylation activity

Sarcosine oxidase (SOX) gene from Thermomicrobium roseum DSM 5159 (GeneBank: ACM06094.1) was inserted into a pMA5 plasmid, expressed in Bacillus subtilis WB600 in soluble form, and then was purified. The purified TrSOX revealed a single band at approximately 43 kDa on 10% reducing SDS-PAGE gel, and was further confirmed by linear trap quadropole mass spectrometry (LTQ-MS) analysis. Regarding CD analysis, the secondary structure of TrSOX was composed of 32.7% α-helix, 17.8% β-sheet, 20.6% β-turn, and 28.5% random coil. Nano-DSC analysis showed that the melting temperature (Tm) and the denaturation enthalpy (ΔH) of TrSOX were 100 ℃ and 552.8 kJ/mol, which indicated that the enzyme should be with outstanding thermo stability. Using N-methylglycine (sarcosine) as substrate, the N-methyl group was depleted and hydrogen peroxide was produced, the Km, kcat and kcat/Km of TrSOX were 61.2 ± 4.3 mmol/L, 3.2 ± 0.2 min−1 and 52.2 L·mol−1 min−1, respectively; additionally, TrSOX showed well adaptation and stability in aqueous/solvent biphasic mixture. Using N-methyltryptophan as substrate, the Km, kcat and kcat/Km of TrSOX were 136.5 ± 7.8 mmol/L, 0.27 ± 0.02 min−1 and 1.98 L·mol−1 min−1, respectively. Additionally, TrSOX showed catalytic activity against other N-methyl or N-ethyl compounds; and for most of the N-methyl-L substrates, the enzyme revealed high chiral selectivity (>100).

Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography-mass spectrometry. Michaelis-Menten kinetic parameters were determined. For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Impact of CYP genotype and inflammatory markers on the plasma concentrations of tramadol and its demethylated metabolites and drug tolerability in cancer patients.

Clinical responses to oral tramadol show a large variation in cancer patients. This study aimed to evaluate the impacts of cytochrome P450 (CYP) genotype and serum inflammatory markers on the plasma concentrations of tramadol and its demethylated metabolites and drug tolerability in cancer patients. The predose plasma concentrations of tramadol and its demethylated metabolites were determined at day 4 or later in 70 Japanese cancer patients treated with oral tramadol. The CYP genotypes, serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels, and the duration of tramadol treatment were evaluated. The CYP2D6 genotype did not affect the plasma tramadol concentration. The plasma concentration of O-desmethyltramadol and its ratio to tramadol were lower in the CYP2D6 intermediate and poor metabolizer (IM + PM) group than in the normal metabolizer (NM) group (P = 0.002 and P = 0.023). The plasma concentration of N-desmethyltramadol and its ratio to tramadol were higher in the CYP2D6 IM + PM group than in the NM group (P = 0.001 and P = 0.001). The CYP2B6*6 and CYP3A5*3 alleles had no effect on the plasma concentrations of tramadol and its demethylated metabolites. The serum IL-6 and CRP levels were inversely correlated with the plasma concentration ratios of N-desmethyltramadol to tramadol and of N,O-didesmethyltramadol to O-desmethyltramadol. The serum IL-6 level was associated with the treatment duration of oral tramadol. The CYP2D6 genotype but not the CYP2B6 and CYP3A5 genotypes affected the plasma concentrations of O- and N-desmethyltramadol through alteration of the tramadol metabolic pathway. The serum IL-6 level was associated with N-demethylation activity and tramadol tolerability.

Substrate Specificity of Human Cytochrome P450 (CYP) 2C Subfamily and Effect of Azole Antifungal Agents on CYP2C8.

The metabolic activities of aminopyrine N-demethylation and tolbutamide methylhydroxylation by the human hepatic cytochrome P450 (P450 or CYP) 2C subfamily were compared and the effects of azole antifungal agent on the drug-metabolizing activity of CYP2C8 were investigated. Aminopyrine N-demethylation and tolbutamide methylhydroxylation by CYP2C8, CYP2C9, and CYP2C19 were determined by the previous reported methods. The effects of five azole antifungal agents, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole, on the aminopyrine N-demethylation activity by CYP2C8 were investigated. With regard to aminopyrine N-demethylation, CYP2C19 had the lowest Michaelis constant (Km) and CYP2C8 had the highest maximal velocity (Vmax) among the CYP2C subfamily members. The Vmax/Km values for CYP2C8 were the highest, followed by CYP2C19. For tolbutamide methylhydroxylation, the Km and Vmax for CYP2C19 were three and six times higher than the corresponding values for CYP2C9, and the Vmax/Km value for CYP2C19 was twice that for CYP2C9, whereas hydroxylated tolbutamide formed by CYP2C8 was not detected. Fluconazole, itraconazole, and voriconazole at a concentration of 2 or 10 µM neither inhibited nor stimulated CYP2C8-mediated aminopyrine N-demethylation activity at substrate concentrations around the Km (5 mM). However, ketoconazole and miconazole noncompetitively inhibited CYP2C8-mediated aminopyrine N-demethylation with the inhibitory constant values of 1.98 and 0.86 µM, respectively. These results suggest that ketoconazole and miconazole might inhibit CYP2C8 clinically.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Status of pyrethroid resistance and mechanisms in Brazilian populations of Tuta absoluta

The tomato leafminer, Tuta absoluta, is a major pest of tomato crops worldwide. This study surveyed the resistance of T. absoluta populations from four regions in Brazil to pyrethroid insecticides, the frequencies of L1014F, T929I and M918T Na channel mutations, and the role of detoxification metabolism in the resistance. Resistance ratios varied from 1- to 11-times among populations and insecticides, but control failure likelihood assays showed that all pyrethroids assessed exhibited no efficacy at all (and thus, 98–100% control failure likelihood) against all T. absoluta populations. The activity of glutathione S-transferase and cytochrome P450-mediated N-demethylation in biochemical assays was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes may play a role in resistance. TaqMan assays were used to screen for the presence of knockdown resistance (kdr) mutations and revealed that the L1014F kdr mutation was fixed in all populations and associated with two super-kdr mutations, M918T and particularly T929I, at high frequency. Altogether, results suggest that control failures are because of mutations in the domain II of the sodium channel, as a prevailing mechanism of resistance to pyrethroids in populations of T. absoluta in Brazil. But, enhanced cytochrome P450-dependent monooxygenases and GST activities also play an important role in the resistance of some populations, which reinforce that pyrethroids must not be used overall to control T. absoluta.

CYP3A4 Intron 6 C>T SNP ( CYP3A4*22 ) Encodes Lower CYP3A4 Activity in Cancer Patients, as Measured with Probes Midazolam and Erythromycin

The CYP3A4*22 allele was recently reported to be associated with reduced CYP3A4 activity. We investigated the impact of this allele on the metabolism of the CYP3A-phenotyping probes, midazolam (MDZ) and erythromycin. Genomic DNA from 108 cancer patients receiving intravenous MDZ and 45 undergoing the erythromycin breath test was analyzed for CYP3A4*22 (rs35599367 C>T) and CYP3A5*3. The MDZ metabolic ratio (1´-OH-MDZ:MDZ) was 20.7% (95% CI: -36.2 to -6.2) lower for CYP3A4*22 carriers compared with CYP3A4*1/*1 patients (p = 0.01). Combining CYP3A4*22 and CYP3A5*3 genotypes showed a 38.7% decrease (95% CI: -50.0 to -27.4; p < 0.001) in 1´-OH-MDZ:MDZ for poor (CYP3A4*22-CYP3A5*3/*3) and 28.0% (95% CI: -33.3 to -22.6; p < 0.001) for intermediate (CYP3A4*1/*1-CYP3A5*3/*3) metabolizers, compared with extensive (CYP3A4*1/*1-CYP3A5*1) CYP3A metabolizers. CYP3A4 erythromycin N-demethylation activity was 40% lower in CYP3A4*22 carriers compared with CYP3A4*1/*1 patients (p = 0.032). The CYP3A4*22 allele is associated with decreased CYP3A4-mediated metabolism, as verified by CYP3A-phenotyping probes.

Disappearance of sexual dimorphism in triptolide metabolism in monosodium glutamate treated neonatal rats

Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), was reported to be sex-dependently metabolized mainly due to sex-related expression of CYP3A2. Sexual dimorphism in the expression of CYP isoforms is affected by sex difference in daily rhythm of growth hormone (GH) secretion. Neonatal administration of monosodium glutamate (MSG) can produce latent developmental defects in GH secretion and associated sex-dependent hepatic enzymes. In the present study, the triptolide metabolism, CYP3A2 expression and CYP3A-dependent activity were evaluated in Sprague-Dawley rats treated neonatally with MSG (4 mg/g) or saline (control) on postnatal days 1, 3, 5, 7 and 9. Treatment with MSG during the neonatal period in both sexes caused a number of disorders characterized by stunted body growth, notable obesity and suppression of GH secretion to barely detectable levels. In addition, neonatal treatment with MSG nearly eliminated the male-specific CYP3A2 expression and significantly reduced the microsomal erythromycin N-demethylation activity in males, while having no effects on CYP3A2 protein in females. Consistent with the P450 findings, the sexual dimorphism of triptolide metabolism completely disappeared in MSG-treated rats. This suggested that neonatal MSG treatment could eliminate the sex-dependent difference in metabolism of triptolide by suppressing CYP3A2 expression and activity in males to the same extent as females.

Cytochrome P450 CYP3A in marsupials: Cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP3A enzymes in marsupials. Given the significant role that CYP3A enzymes play in the metabolism of both endogenous and exogenous compounds, the clone provides an important step in elucidating the metabolic capacity of marsupials.

In-vitro metabolism of YM17E, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, by liver microsomes in man.

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl) ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [14C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6 beta-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and alpha-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.

Species Difference between Cynomolgus Monkeys and Humans on Cytochromes P450 2D and 3A-Dependent Drug Oxidation Activities in Liver Microsomes

Cynomolgus monkeys [Macaca fascicularis (mf)] are widely used to determine pharmacokinetics and toxicological potential of many drug candidates as human models in the drug discovery and development. Cytochrome P450s (P450, CYP), one of the most important enzymes in drug metabolism, in monkey livers are generally similar to corresponding human P450s exhibiting high degrees of homologies in cDNAs and amino acid sequences. Species differences regarding important liver P450 3A and 2D function were examined between cynomolgus monkeys and humans using typical human P450 probe reactions using midazolam (a P450 3A marker), dextromethorphan and bufuralol (P450 2D markers). P450 3A-mediated midazolam 1'-hydroxylation activities in liver microsomes from individual monkey were highly correlated with midazolam 4'-hydroxylation activities but not correlated with dextromethorphan N-demethylation activities. Recombinant monkey CYP2D17 and CYP2D44 catalyzed dextromethorphan O- and N-demethylations as well as monkey mfCYP3A4 and mfCYP3A5 did. On the other hand, contributions of corresponding P450 2D6 and P450 3A4/5 to dextromethorphan N- and O-demethylations, in human liver microsomes were negligible under the present conditions. From these results, monkey P450 2D and 3A enzymes might have broader substrate specificity toward dextromethorphan oxidation than those in human livers. Special attention should be paid when enzymatic and pharmacokinetic data are extrapolated from monkeys to humans.

Influence of intravenous methylprednisolone pulse treatment on the disposition of ciclosporin and hepatic CYP3A activity in rats.

We examined the effects of high-dose methylprednisolone (MP) on the disposition of ciclosporin (CsA) and hepatic microsomal CYP3A activity using rats. Methylprednisolone sodium succinate (MPS), a prodrug of MP, was intravenously administered as repeated doses (66.3 mg kg(-1)) for 3 days or as a single dose. In MP-treated rats, a significant increase was observed in the total body clearance (CL(tot)) and elimination rate constant (Ke) of intravenously administered CsA. The enzyme activities of triazolam hydroxylations and erythromycin N-demethylation in hepatic microsomes were also enhanced by about 50% by MP treatment, suggesting that the alteration in the CsA pharmacokinetics was due to significant induction of the hepatic CYP3A responsible for the metabolic conversion of CsA. In contrast, no significant changes in the values of CL(tot) and Ke were found following a single treatment with MP. On the other hand, MP inhibited the CYP3A-mediated triazolam hydroxylations in a concentration-dependent manner. The difference between the in-vivo and in-vitro inhibitory behaviours of MP was attributed to the rapid elimination of MP after biotransformation from MPS because the plasma MP concentration decreased with a half-life of 15 min immediately after reaching a level close to the inhibition constant for the triazolam 4-hydroxylation reaction (32.4 microM). Although there is a general consideration that MP cannot act as an enzyme inducer at maintenance doses, the present results strongly suggest that high-dose MP is likely to interact pharmacokinetically with CsA by inducing hepatic CYP3A. These results may provide basic explanations for the clinical experience that blood CsA levels are reduced during MP pulse therapy.

Induction of Hepatic Cytochrome P450s by the Herbal Medicine Sophora flavescens Extract in Rats: Impact on the Elimination of Theophylline

The roots of Sophora flavescens (Sf) have been widely used as a herbal medicine for the treatment of diarrhea, gastrointestinal hemorrhage, and eczema. Cytochrome P450 (P450) forms including CYP1A2, CYP2B, CYP2E1, and CYP3A participate in the oxidative metabolism of theophylline, which is an important bronchodilation agent with a narrow therapeutic index. To assess the interaction of Sf with theophylline, the effects of Sf extract on theophylline-metabolizing P450s and on the pharmacokinetic profile of theophylline were investigated in male Sprague-Dawley rats. Oral treatment of rats with the Sf extract caused dose-dependent increases of liver microsomal oxidation activities toward 7-ethoxyresorufin, 7-pentoxyresorufin, and nifedipine. However, nitrosodimethylamine N-demethylation activity was not affected. The ingestion of Sf extract stimulated theophylline 8-oxidation and N-demethylation activities. The increases of oxidative activities were in consensus with the elevation of the protein levels of CYP1A2, CYP2B1/2, CYP2C11, and CYP3A. Sf-treatment increased the clearance of theophylline and decreased the area under the concentration-time curve (AUC) and the area under the moment curve (AUMC). These results demonstrate that Sf reduces blood theophylline concentration through facilitating the elimination of theophylline. In patients taking Sf, possible P450 induction-induced drug interaction should be noted to decrease the risk of therapeutic failure or adverse effects resulting from the use of additional therapeutic agents.

The Nanoparticle Photogenerated by Association of Phenothiazine Nucleus to Poly(ethylene glycol) Protects Photodamage in Mitochondrial Membrane Unsaturated Lipids

This study investigated the hypothesis that human term placental lipoxygenase (HTPLO) and soybean lipoxygenase (SLO) are capable of mediating N-demethylation of selected phenothiazines and insecticides in the presence of linoleic acid (LA).In addition to being LA dependent, the N-demethylation reaction mediated by HTPLO and SLO was limited by incubation time, pH of the medium, concentration of the enzyme and the substrate. Using Nash reagent to monitor formaldehyde production, the specific activity for LA-dependent N-demethylation of chlorpromazine, a model phenothiazine, was determined to be 1.7±0.3 nmoles/min/mg HTPLO. Besides chlorpromazine, N-demethylation of promazine, promethazine and trimeprazine was also observed. The insecticide, aminocarb, displayed a specific activity of 2.2±0.3 nmoles/min/mg HTPLO for N-demethylation. Other insecticides, namely chlordimeform, dicrotophos and zectran, were oxidized in a similar manner. As compared with HTPLO, the rates of N-demethylation of phenothiazines and insecticides mediated by SLO were higher. Classical inhibitors of lipoxygenase, as well as antioxidants and free radical scavengers, caused a dose-dependent reduction in the production of formaldehyde from chlorpromazine and aminocarb by HTPLO. These results clearly demonstrate the ability of polyunsaturated free fatty acids to support N-demethylation of xenobiotics via the lipoxygenase pathway.

Diazepam Metabolism in the Kidneys of Male and Female Rats of Various Strains

Previously, we have reported drastic strain differences of diazepam metabolism in the livers of a variety of rat strain. In this study, to characterize strain and sex differences of diazepam metabolism in the kidney, renal microsomal diazepam metabolic activities were determined in the Dark Agouti (DA), Sprague-Dawley (SD), Brown Norway (BN) and Wistar (WS) strains of rat. We found that the major pathway of diazepam metabolism in the kidney was diazepam N-demethylation, which is different from that in the liver, 3-hydroxylation. A Dose-course (12.5-200 muM of diazepam) study revealed that the DA and WS male rats had higher diazepam N-demethylation activity than the SD and BN rats. In contrast to the males, a lower activity of diazepam N-demethylation was observed in female BN rats. By Western blot analysis, constitutive protein expressions of cytochrome P450 (CYP) 2C11, which is responsible for diazepam N-demethylation, were detected in the 4 strain in both the male and female rats, and the BN rats had lower expression levels of CYP2C11 protein. However, we did not observe significant differences in the kinetic parameters of diazepam N-demethylation. Our results suggested that there was a strain difference in CYP-dependent diazepam N-demethylation in the rat kidney, which is different from the finding in liver microsomes.

Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be -21 cm3mol(-1), which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.

Induction of Cytochrome P450 CYP3A by St John's Wort in the Rat Liver and Intestine

It has been well reported that complementary medicines can significantly alter the way the body handles conventional drugs, leading to potential fatal herb-drug interactions. The aim of the present study was to investigate the molecular mechanism of drug interactions involving St John's wort (SJW) (Hypericum perforatum L), a popular herbal medicine widely used for depression, particularly examining changes in the expression of cytochrome P450 CYP3A, the most abundant drug metabolising CYP enzymes in man.Eighteen Sprague-Dawley (SD) rats were assigned randomly into 3 groups (n = 6/group): control, low dose and high dose (500 and 1000 mg/kg/day of SJW, equal to 1500 and 3000 µg/kg/day of Hypericin). Each group was treated with SJW or control preparation, by gastric gavage, for 14 consecutive days. Liver and intestinal CYP3A activity and protein and mRNA levels, from five segments of the intestine, were examined using CYP3A-dependent erythromycin N-demethylation activity assay, quantitative immuno-blotting and real-time RT-PCR.Increase in CYP3A activity and protein level by SJW was observed in some intestinal regions, with a 3.0 fold increase in liver CYP3A activity and a 10.6 fold increase in liver CYP3A1 mRNA (p &lt; 0.05) in a dose dependent manner. The results suggested that up regulation of liver CYP3A mRNA and differential induction of intestinal CYP3A play an important role in the molecular mechanism of herb-drug interactions.

Identification of Human Cytochrome P450 Isozymes Involved in Diphenhydramine N-Demethylation

Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isozymes that mediate the metabolism of diphenhydramine in the range of clinically relevant concentrations (0.14-0.77 microM) remain unclear. Therefore, P450 isozymes involved in N-demethylation, a main metabolic pathway of diphenhydramine, were identified by a liquid chromatography-mass spectrometry method developed in our laboratory. Among 14 recombinant P450 isozymes, CYP2D6 showed the highest activity of diphenhydramine N-demethylation (0.69 pmol/min/pmol P450) at 0.5 microM. CYP2D6 catalyzed diphenhydramine N-demethylation as a high-affinity P450 isozyme, the K(m) value of which was 1.12 +/- 0.21 microM. In addition, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in diphenhydramine N-demethylation was confirmed by using P450 isozyme-specific inhibitors. In addition, contributions of these P450 isozymes estimated by the relative activity factor were in good agreement with the results of inhibition studies. Although an inhibitory effect of diphenhydramine on the metabolic activity of CYP2D6 has been reported previously, the results of the present study suggest that it is not only a potent inhibitor but also a high-affinity substrate of CYP2D6. Therefore, it is worth mentioning that the sedative effect of diphenhydramine might be caused by coadministration of CYP2D6 substrate(s)/inhibitor(s). In addition, large differences in the metabolic activities of CYP2D6 and those of CYP1A2, CYP2C9, and CYP2C19 could cause the individual differences in anti-allergic efficacy and the sedative effect of diphenhydramine.

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.