Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The major metabolites of tamoxifen (tam) formed by animal and human liver microsomes are mono-N-demethylated tam, 4-hydroxy-tam (4-OH-tam), and tam-N-oxide. The N-desmethylated-tam and 4-OH-tam are formed by P450s, whereas the N-oxide is primarily formed by flavin-containing monooxygenase. Because 4-OH-tam is a highly potent antiestrogen (and possibly is the active anticancer tam metabolite) and is on the path of formation of the reactive intermediate that binds covalently to proteins and DNA, it was of importance to identify the P450(s) catalyzing its formation. In the current study, three different preparations of expressed human P450s in Escherichia coli, lymphoblastoma cells, and insect cell line and livers from several human donors were used to identify the P450 isoform catalyzing the 4-hydroxylation (preliminary results were reported by Dehal et al., Eleventh International Symposium on Microsomes and Drug Oxidations, p. 71. Los Angeles, 1996). Tam metabolism was examined with human CYP2C8, 2C9, 2C18, 2C19, and 2D6 expressed in E. coli. These P450s were reconstituted with P450 reductase and lipid and were incubated with 50 microM [3H]tam and NADPH at 37 degrees C for 60 min. Essentially all of the recombinant P450s catalyzed the N-demethylation to various degrees; however, only 2D6 yielded detectable levels of 4-OH-tam. The inclusion of cytochrome b5 in the reconstituted system of 2D6 and 2C9 did not significantly affect the rate of 4-hydroxylation, indicating that b5 is not essential for this activity. Tam metabolism by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4, expressed in lymphoblastoma cells, revealed that only 2D6 significantly catalyzed the 4-hydroxylation. Tam metabolism by CYP2D6 coexpressed with P450 reductase in a baculovirus infected insect cell line ("supersomes") exhibited marked tam 4-hydroxylation. In an experiment with human liver microsomes, the inclusion of quinidine, a specific 2D6 inhibitor, resulted in approximately 50% inhibition of tam 4-hydroxylation without affecting N-demethylation. Polyclonal antibodies raised against 2D6 moderately inhibited (approximately 30%) the 4-hydroxylation in human liver microsomes. These results demonstrate a significant contribution by CYP2D6 to the catalysis of tam-4-hydroxylation by human liver.
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PMID:CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver. 927 5

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer prodrugs that are bioactivated in the liver by specific cytochrome P450 enzymes (CYPs). The therapeutic activity of these antitumor agents can be compromised by a low therapeutic index that is, in part, due to the systemic distribution of activated drug metabolites. Here, recombinant retroviruses were used to deliver six different CPA- or IFA-metabolizing human CYP genes to 9L gliosarcoma cells: 2B6, 2C8, 2C9, 2C18 (Met385 and Thr385 alleles), 2C19, and 3A4. Intratumoral cytochrome P450 expression conferred substantial sensitivity to CPA cytotoxicity, with the most dramatic effects seen with CYP2B6. Strong CPA chemosensitivity was also seen following transduction of CYP2C18-Met, despite a very low level of CYP protein expression (>60-fold lower than that of 2B6). In contrast to CPA, the cytotoxicity of IFA was greatest toward tumor cells transduced with CYP3A4, followed by CYPs 2B6 and 2C18-Met. A substantial further increase in chemosensitivity was achieved upon transduction of 2B6 or 2C18-Met-expressing tumor cells with P450 reductase, which provided for more efficient intratumoral prodrug activation and cytotoxicity at lower drug concentrations. With 2B6- plus P450 reductase-transduced tumor cells, CPA but not IFA conferred a strong cell contact-independent bystander cytotoxic effect on non-P450-expressing 9L cells. CPA treatment of tumors that were transduced with 2B6 or 2C18-Met together with P450 reductase and were grown s.c. in immunodeficient mice resulted in a large enhancement of the liver P450-dependent antitumor effect seen with control 9L tumors, with no apparent increase in host toxicity (growth delay of >25-50 days in P450-expressing tumors versus approximately 5-6 days without P450). CYP2B6 plus P450 reductase and CYP2C18-Met plus P450 reductase thus appear to be excellent gene combinations for use with CPA in P450/prodrug activation-based cancer gene therapy.
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PMID:Retroviral transfer of human cytochrome P450 genes for oxazaphosphorine-based cancer gene therapy. 976 69

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the Km values by these three P450 enzymes were determined to be 11.0, 105, and 132 microM, respectively, and the Vmax values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 microM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 microM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 microM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 microM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 microM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 microM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes.
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PMID:Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. 1035 Jan 85

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a,l]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450s (but not CYP1B1) (in T. ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.
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PMID:Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. 1150 24

Amino terminal sequence modification of cytochrome P450 enzymes is often necessary to achieve expression in bacteria. The aim of this study was to examine the effect of such modifications on membrane integration and P450 activity. Forms that retained substantial N-terminal hydrophobic sequences remained unaffected by treatments to remove peripheral membrane proteins and were released only by detergent. Truncated P450s 2A13, 2C9 (delta 3-20), 2C19 (delta 3-20), 2D6 (DB11) and 2E1 remained principally membrane-bound, but some P450 was found in the soluble fraction and could be partially extracted by alkaline and high salt treatments. The subcellular localization of P450s 2C9 and 2C19 assessed by fluorescence microscopy mirrored the distribution between subcellular fractions. The MALLLAVFL modified forms of P450 2C9 YFP, P450 2C18 YFP and P450 2C19 YFP were found primarily at the periphery of the cells, whereas the truncated forms of P450 2C9 (delta 3-20) YFP and 2C19 (delta 3-20) YFP were observed at the periphery as well as inside the cells. N-terminal variants of P450s 2C9 and 2C19 showed altered kinetics towards form-selective substrates. Rates of diclofenac 4 -hydroxylation by P450 2C9 and luciferin H-EGE metabolism by P450 2C19 were higher for the MALLLAVFL-modified forms compared with the (delta 3-20) truncated forms despite supplementation of truncated form incubations with additional reductase. Thus, N-terminal sequence modifications changed the degree of membrane integration, potentially affecting subcellular localization, interactions with redox partners, and hence enzymatic activity.
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PMID:Membrane integration of recombinant human P450 forms. 1953 86