RESUMEN
Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.
Asunto(s)
Anticarcinógenos/farmacología , Anticarcinógenos/farmacocinética , Inhibidores del Citocromo P-450 CYP2E1 , Isotiocianatos/farmacología , Isotiocianatos/farmacocinética , Biotransformación , Humanos , Técnicas In Vitro , Isocianatos/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Oxidación-ReducciónRESUMEN
1. UDP-glucuronosyltransferases (UGTs) are a group of drug-metabolizing enzymes that catalyse the conjugation of endogeonous compounds and xenobiotics to yield hydrophilic glucuronides which subsequently undergo excretion. This report describes an approach for the identification and accurate quantitation of human UGT1A1 in complex biological matrices using liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis of protein digests. 2. A stable isotope-labelled (SIL) peptide of a unique peptide spanning residues 54-69 in exon 1 of the human UGT1A1 protein with the sequence RIYLSADPALVVIEHG was synthesized. The peptide sequence synthesized was in the reverse order of the human peptide with the stable isotope-labels in the amino acid arginine ((13)C6(15)N4) resulting in an increase in the mass of the SIL peptide of 10 amu, from 1753 to 1763. The SIL peptide was quantitated by injecting increasing concentrations of the peptide into the LC-MS to obtain a standard curve. 3. The labelled peptide along with precursor ion monitoring was used to quantify the levels of UGT1A1 in commercial recombinant preparations (supersomes) and individual human liver microsomal samples and pooled human liver micrsomes obtained from BD Biosciences. 4. Glucuronidation activity studies were performed, which demonstrated a positive correlation between enzyme activity levels and the UGT1A1 content in the liver microsomes obtained from individual human donors.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Marcaje Isotópico , Espectrometría de Masas/métodos , Microsomas Hepáticos/enzimología , Péptidos/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Calibración , Cromatografía Liquida , Estradiol/metabolismo , Glucurónidos/metabolismo , Humanos , Datos de Secuencia Molecular , Péptidos/química , Tripsina/metabolismoRESUMEN
The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide ¹88FHYQDQE¹94 with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.
Asunto(s)
Anticarcinógenos/farmacocinética , Apoproteínas/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Indolquinonas/farmacocinética , Oxidorreductasas N-Desmetilantes/metabolismo , Tamoxifeno/farmacocinética , Anticarcinógenos/farmacología , Antineoplásicos Hormonales/farmacocinética , Antineoplásicos Hormonales/farmacología , Biotransformación , Citocromo P-450 CYP2B6 , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Hidroxilación , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Tamoxifeno/farmacologíaRESUMEN
Selegiline, the R-enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6. The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation (7-EFC) activity of CYP2B6 as well as bupropion metabolism. The inactivations were time-, concentration-, and NADPH-dependent and were characterized by K(I) values of 0.14 and 0.6 µM, k(inact) values of 0.022 and 0.029 min⻹, and t(½) values of 31.5 and 24 min, respectively. In standard inhibition assays, selegiline increased the K(m) of CYP2B6 for bupropion from 10 to 92 µM and decreased the k(cat) by â¼50%. The reduced carbon-monoxide difference spectrum revealed over a 50% loss in the cytochrome P450 spectrum in the inactivated sample, with no loss in heme, and there was â¼70% loss in enzyme activity. Trapping of the reactive metabolite using GSH led to the identification of a GSH-selegiline conjugate with a m/z 528 that could be explained by hydroxylation of selegiline followed by the addition of glutathione to the propargyl moiety after oxygenation to form the ketene intermediate. Liquid chromatography-tandem mass spectrometry analysis of the labeled protein following digestion with trypsin revealed the peptide 64DVFTVHLGPR7³ as the peptide modified by the reactive metabolite of selegiline and the site of adduct formation is Asp64.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Bupropión/metabolismo , Glutatión/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Péptidos/metabolismo , Selegilina/farmacología , Secuencia de Aminoácidos , Antidepresivos de Segunda Generación/metabolismo , Antiparkinsonianos/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Cromatografía Liquida/métodos , Citocromo P-450 CYP2B6 , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Escherichia coli/metabolismo , Hemo/metabolismo , Humanos , Hidroxilación/efectos de los fármacos , Datos de Secuencia Molecular , NADP/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the K(m) values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype-phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Antidepresivos de Segunda Generación/farmacocinética , Hidrocarburo de Aril Hidroxilasas/genética , Benzoxazinas/metabolismo , Bupropión/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Alquinos , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Células COS , Catálisis , Chlorocebus aethiops , Cumarinas/metabolismo , Ciclopropanos , Citocromo P-450 CYP2B6 , Transporte de Electrón , Escherichia coli/metabolismo , Compuestos Férricos/metabolismo , Variación Genética , Cinética , Mutagénesis , Mutación/fisiología , Oxidorreductasas N-Desmetilantes/metabolismo , Polimorfismo Genético , terc-Butilhidroperóxido/metabolismoRESUMEN
Anandamide is an arachidonic acid-derived endogenous cannabinoid that regulates normal physiological functions and pathophysiological responses within the central nervous system and in the periphery. Several cytochrome P450 (P450) isoforms metabolize anandamide to form hydroxylated and epoxygenated products. Human CYP2B6 and CYP2D6, which are expressed heterogeneously throughout the brain, exhibit clinically significant polymorphisms and are regulated by external factors, such as alcohol and smoking. Oxidative metabolism of anandamide by these two P450s may have important functional consequences for endocannabinoid system signaling. In this study, we investigated the metabolism of anandamide by wild-type CYP2B6 (2B6.1) and CYP2D6 (2D6.1) and by their common polymorphic mutants 2B6.4, 2B6.6, 2B6.9, and 2D6.34. Major differences in anandamide metabolism by the two isoforms and their mutants were found in vitro with respect to the formation of 20-hydroxyeicosatetraenoic acid ethanolamide (20-HETE-EA) and 14,15-epoxyeicosatetraenoic acid ethanolamide (14,15-EET-EA). Pharmacological studies showed that both 20-HETE-EA and 14,15-EET-EA bind to the rat brain cannabinoid CB1 receptor with lower affinities relative to that of anandamide. In addition, both products are degraded more rapidly than anandamide in rat brain homogenates. Their degradation occurs via different mechanisms involving either fatty acid amide hydrolase (FAAH), the major anandamide-degrading enzyme, or epoxide hydrolase (EH). Thus, the current findings provide potential new insights into the actions of inhibitors FAAH and EH, which are being developed as novel therapeutic agents, as well as a better understanding of the interactions between the cytochrome P450 monooxygenases and the endocannabinoid system.
Asunto(s)
Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Moduladores de Receptores de Cannabinoides/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Epóxido Hidrolasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Animales , Ácidos Araquidónicos/química , Ácidos Araquidónicos/farmacocinética , Hidrocarburo de Aril Hidroxilasas/genética , Encéfalo/metabolismo , Moduladores de Receptores de Cannabinoides/química , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2D6/genética , Endocannabinoides , Epóxido Hidrolasas/antagonistas & inhibidores , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hidroxilación , Masculino , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/genética , Polimorfismo Genético , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
17-alpha-Ethynylestradiol (17EE) is a mechanism-based inactivator of P450 2B1 and P450 2B6 in the reconstituted monooxygenase system. The loss in enzymatic activity was due to the binding of a reactive intermediate of 17EE to the apoprotein. P450 2B1 and P450 2B6 were inactivated by 17EE and digested with trypsin. The peptides obtained following digestion with trypsin of 17EE-inactivated P450 2B1 and P450 2B6 were separated by liquid chromatography and analyzed by ESI-MS. Adducted peptides exhibiting an increase in mass consistent with the addition of the mass of the reactive intermediate of 17EE were identified for each enzyme. Analysis of these modified peptides by ESI-MS/MS and precursor ion scanning facilitated the identification of the Ser360 in both enzymes as a site that had been adducted by a reactive intermediate of 17EE. A P450 2B1 mutant where Ser360 was replaced by alanine was constructed, expressed, and purified. Activity and inactivation studies indicated that mutation of the Ser360 residue to alanine did not prevent inactivation of the mutant enzyme by 17EE. These observations suggest that Ser360 is not critical for the catalytic function of these P450s. Spectral binding studies of the 17EE-inactivated P450 2B1 and P450 2B6 indicated that modification of the enzymes by the reactive intermediate of 17EE resulted in an enzyme that was no longer capable of binding substrates. These results suggest that the inactivation by 17EE may be due to modification of an amino acid residue in the substrate access channel near the point of entry into the active site.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Etinilestradiol/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Secuencia de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/química , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B6 , Activación Enzimática , Datos de Secuencia Molecular , Mutación/genética , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/química , Serina/genética , Serina/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Testosterona/metabolismo , Tripsina/metabolismoRESUMEN
Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar K(m) values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Citocromo P-450 CYP2D6/fisiología , Alcamidas Poliinsaturadas/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidónico/metabolismo , Encéfalo/metabolismo , Endocannabinoides , Compuestos Epoxi/metabolismo , Humanos , Hidroxilación , Microsomas/metabolismo , Mitocondrias/metabolismo , Factores de TiempoRESUMEN
The abilities of 7-coumarin propargyl ether (CPE) and 7-(4-trifluoromethyl)coumarin propargyl ether (TFCPE) to act as mechanism-based inactivators of P450 3A4 and 3A5 in the reconstituted system have been investigated using 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and testosterone as probes. CPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner characteristic of a mechanism-based inactivator with a half-maximal inactivation (K(I)) of 112 microM, a maximal rate of inactivation (k(inact)) of 0.05 min(-1), and a t(1/2) of 13.9 min. Similarly, TFCPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner with a K(I) of 14 microM, a k(inact) of 0.04 min(-1), and a t(1/2) of 16.5 min. Parallel losses of P450 3A4 enzymatic activity and heme were observed with both compounds as measured by high-performance liquid chromatography and reduced CO spectra. Interestingly, neither compound inhibited the BFC O-debenzylation activity of P450 3A5. Reactive intermediates of CPE and TFCPE formed by P450 3A4 were trapped with glutathione, and the resulting adducts were identified using tandem mass spectral analysis. Metabolism studies using TFCPE resulted in the identification of a single metabolite that is formed by P450 3A4 but not by P450 3A5 and that may play a role in the mechanism-based inactivation.
Asunto(s)
Alquinos/síntesis química , Cumarinas/síntesis química , Inhibidores del Citocromo P-450 CYP3A , Éteres/síntesis química , Imidazoles/síntesis química , Alquinos/metabolismo , Alquinos/farmacología , Cumarinas/metabolismo , Cumarinas/farmacología , Citocromo P-450 CYP3A/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Éteres/metabolismo , Éteres/farmacología , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismoRESUMEN
It has been demonstrated previously that several 3-trifluoromethyl-3-(4-alkoxyphenyl)diaziridines inhibit the 7-ethoxy-4-(trifluoroethyl)coumarin (7-EFC) O-deethylation activity of P450 2B6 in a mechanism-based manner. In contrast, 3-trifluoromethyl-3-(4-methylthio)phenyl)diaziridine did not have any effect on the activity of P450 2B6. It is interesting that both the alkoxy and the thiophenyl compounds were metabolized by P450 2B6. In this report, the structure-activity relationships for the mechanism-based inactivation of cytochrome P450 2B6 by a series of aryl diaziridines were investigated. Three diaziridines that did not contain a 4-alkoxy-substituent on their phenyl ring, namely, 3-trifluoromethyl-3-(3-methoxyphenyl)diaziridine, 3-trifluoromethyl-3-phenyl diaziridine, and 3-trifluoromethyl-3-(4-chlorophenyl)diaziridine had no effect on the P450 2B6 7-EFC activity. Another analog that did not contain a diaziridine substructure, 3-trifluoromethyl-3-(4-methoxyphenyl)ethanone, also had no effect on the activity of P450 2B6. Glutathione ethyl ester adducts of the phenyldiaziridine reactive intermediates were isolated from reaction mixtures of the inactivated samples and analyzed by liquid chromatography-tandem mass spectrometry. The structures of the conjugates suggested that the electrophilic reactive intermediate in each case was a quinone methide (quinomethane), 4-ethylidene-cyclohexa-2,5-dienone, generated from the 4-alkoxyphenyldiaziridines by removal of both of the diaziridine and the 4-alkyl groups. In conclusion, the determinant factor for the mechanism-based inactivator activity of the aryl diaziridines seems to be the formation of the reactive quinomethane intermediate, which is generated from the 4-alkoxyphenyl diaziridines by a cytochrome P450-catalyzed metabolic reaction.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Aziridinas/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Aziridinas/síntesis química , Aziridinas/química , Cumarinas/metabolismo , Citocromo P-450 CYP2B6 , Glutatión/análogos & derivados , Glutatión/metabolismo , Técnicas In Vitro , Oxidorreductasas N-Desmetilantes/metabolismo , Relación Estructura-ActividadRESUMEN
The metabolism of arylhydrazines by cytochromes P450 (P450s) has previously been shown to yield aryl-iron complexes that inhibit P450 enzymes as a result of heme modification. These modifications of the heme have been used to probe the topology of the active site of several P450s. Therefore, diaziridines containing one or more substitutions on the phenyl ring were synthesized and evaluated as potential mechanism-based inactivators of P450 2B enzymes that could be used to elucidate the active site topology. Five of the six trifluoroaryldiaziridines tested selectively inactivated P450 2B6 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner as measured using the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation assay. The kinetic parameters for P450 2B6 inactivation by the five compounds were calculated. Analysis of the P450 heme from P450s inactivated by the five substituted diaziridines suggested that the activity loss was not due to heme destruction as measured by the reduced-CO spectrum or high-performance liquid chromatography of the P450 heme. Dialysis experiments indicated the irreversible nature of the inactivation and the reaction between the diaziridine compounds and the P450 enzyme. Interestingly, a thiomethyl-substituted phenyl diaziridine had no effect on the activity of P450 2B6 in the reconstituted system, but competitively inhibited the O-debenzylation activity of P450 3A4 with 7-benzyloxy-4-(trifluoromethyl)coumarin as substrate. Binding spectra suggest that this compound bound reversibly to P450 2B6, and preliminary results indicate that 3-(4-methylthiophenyl)-3-(trifluoromethyl)diaziridine is metabolized by P450 2B6.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Aziridinas , Inhibidores Enzimáticos , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Aziridinas/síntesis química , Aziridinas/química , Aziridinas/farmacología , Citocromo P-450 CYP2B6 , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Estructura Molecular , NADP/química , Relación Estructura-Actividad , Factores de TiempoRESUMEN
We have previously observed that the quadruple (S407T-N417D-A419T-K473M) and triple (S407T-N17D-A419T) mutants of the chimeric construct of P450 2B1/2B2 do not undergo mechanism-based inactivation by 17alpha-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether (tBMP). The ability of these mutants to metabolize 17EE, benzphetamine, and testosterone has been investigated. The profile for 17EE metabolism by both mutants was characteristic of both wild-types. The two mutants metabolized testosterone to form androstenedione with no formation of the hydroxy products as was seen with both the wild-types. Benzphetamine metabolism by the mutants showed that both mutants exhibited an increased tendency to catalyze demethylation rather than debenzylation. In the presence of the alternate oxidants cumene hydroperoxide and tert-butyl hydroperoxide, the wild-type 2B1 was not inactivated by 17EE. Metabolism of 17EE by 2B1 supported by these alternate oxidants revealed differences in the metabolites that may be related to the inability of 2B1 to be inactivated under these conditions.
Asunto(s)
Ácido Aspártico/química , Citocromo P-450 CYP2B1/química , Treonina/química , Ácido Aspártico/genética , Benzfetamina/metabolismo , Citocromo P-450 CYP2B1/genética , Etinilestradiol/metabolismo , Oxidantes/metabolismo , Testosterona/metabolismo , Treonina/genéticaRESUMEN
The polymorphic human cytochrome P450 (P450) 2B6 is primarily responsible for the metabolism of several clinically relevant drugs including bupropion, cyclophosphamide, propofol, and efavirenz. Although a number of single nucleotide polymorphisms have been found in the P450 2B6 gene, the influence of these variants on the metabolism of substrates and on the response to known inactivators of P450 2B6 has not been examined. We have compared the metabolism of different substrates of P450 2B6 (P450 Delta2B6) and the effects of mechanism-based inactivators with that observed with the polymorphic P450 Delta2B6 K262R in a reconstituted monooxygenase system (reconstituted system). Metabolism of bupropion by P450 Delta2B6 K262R resulted in increased production of hydroxybupropion compared with P450 Delta2B6. However, production of formaldehyde from the metabolism of benzphetamine by the P450 Delta2B6 K262R mutant was significantly less than that of the wild-type isozyme. P450 Delta2B6 K262R formed fewer benzphetamine metabolites compared with the wild type. N,N',N''-Triethylenethiophosphoramide (tTEPA) and bergamottin decreased the ability of both enzymes to hydroxylate bupropion and to O-deethylate 7-hydroxy-4-(trifluoromethyl)coumarin (7-EFC). Incubation with 17-alpha-ethynylestradiol decreased bupropion hydroxylation and 7-EFC O-deethylation with the wild-type enzyme but had no effect on the mutant. The kinetics for inactivation of the variant by tTEPA and bergamottin were determined using 7-EFC. The KI values for inactivation of the variant were significantly greater than those determined for the wild-type enzyme. These data demonstrate a functional difference between P450 Delta2B6 and the allelic variant P450 Delta2B6 K262R.
Asunto(s)
Alelos , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Variación Genética/genética , Mutación , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Citocromo P-450 CYP2B6 , Relación Dosis-Respuesta a Droga , Humanos , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Especificidad por Sustrato/genéticaRESUMEN
The homologous rat cytochrome P450s 2B1 and 2B2 differ by 13 amino acids. A chimeric construct of P450 2B1/2B2 was used in conjunction with several site-directed mutants to identify key residues involved in the inactivation of P450 2B1 by two acetylenic compounds, 17alpha-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether (tBMP). 17EE is a mechanism-based inactivator of P450 2B1 but not of P450 2B2. We show here that tBMP is also a mechanism-based inactivator of P450 2B1 and not P450 2B2. Minimal loss in 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) activity was observed when P450 2B1 G478A was incubated with either inactivator, suggesting that this residue plays a role in the inactivation. However, P450 2B2 A478G behaved like wild-type P450 2B2, indicating that this residue alone is not sufficient for inactivation. A chimeric construct of P450 2B1/2B2 that is essentially P450 2B1 with five residues of P450 2B2 (including residue 478), was not inactivated by either tBMP or 17EE, suggesting that these five residues are important for inactivation. Sequential mutagenesis of the chimeric construct to quadruple (S407T-N417D-A419T-G478A) and triple (S407T-N417D-A419T) mutants of P450 2B1 did not result in inactivation by either inactivator. However, the triple mutant with mutations only in non-substrate recognition site (SRS) regions still exhibits wild-type P450 2B1 7-EFC O-deethylation activity with a K(m) value of 25 microM and V(max) of 8 nmol/min/nmol P450. These results demonstrate that substitution of three non-SRS residues in P450 2B1 leads to protection against inactivation of 2B enzymes by these two acetylenic compounds.
Asunto(s)
Alquinos/farmacología , Aminoácidos/metabolismo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Éteres/farmacología , Etinilestradiol/farmacología , Aminoácidos/aislamiento & purificación , Animales , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Hemo/metabolismo , Mutación , Ratas , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Silybin, a major constituent of the milk thistle, is used to treat several liver disorders. Silybin inactivated purified, recombinant cytochromes P450 (P450) 3A4 and 2C9 in a mechanism-based manner. The inactivations were time-, concentration-, and NADPH-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl-)coumarin O-debenzylation activity (P450 3A4) was characterized by a K(I) of 32 microM, a k(inact) of 0.06 min(-1), and a t(1/2) of 14 min. Testosterone metabolism to 6-beta-hydroxytestosterone (P450 3A4) was also inactivated with a K(I) of 166 microM, a k(inact) of 0.08 min(-1), and a t(1/2) of 9 min. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified human P450 2C9 was inactivated with a K(I) of 5 microM, a k(inact) of 0.14 min(-1), and a t(1/2) of 7 min. Parallel loss of heme was observed with both P450s. Activity of both P450 enzymes was not recovered after removal of silybin either by dialysis or by spin gel filtration. In addition, silybin inhibited the glucuronidation of 7-hydroxy-4-trifluoromethylcoumarin catalyzed by recombinant hepatic UDP-glucuronosyltransferases (UGTs) 1A1, 1A6, 1A9, 2B7, and 2B15, with IC(50) values of 1.4 microM, 28 microM, 20 microM, 92 microM, and 75 microM, respectively. Silybin was a potent inhibitor of UGT1A1 and was 14- and 20-fold more selective for UGT1A1 than for UGT1A9 and UGT1A6, respectively. Thus, careful administration of silybin with drugs primarily cleared by P450s 3A4 or 2C9 is advised, since drug-drug interactions cannot be excluded. The clinical significance of in vitro UGT1A1 inhibition is unknown.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450 , Glucuronosiltransferasa/antagonistas & inhibidores , Silimarina/farmacología , Hidrocarburo de Aril Hidroxilasas/aislamiento & purificación , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Glucuronosiltransferasa/metabolismo , Hemo/metabolismo , Cinética , NADP , Extractos Vegetales/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Silibina , Testosterona/metabolismoRESUMEN
Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.