RESUMEN
A highly conserved threonine in the I-helix of cytochrome P450s has been suggested to play an important role in dioxygen activation, a critical step for catalytic turnover. However, subsequent studies with some P450s in which this highly conserved threonine was replaced by another residue such as alanine showed that significant catalytic activities were still retained when the variants were compared with the wild type enzymes. These results make the role of this residue unclear. We provide data here that suggest a novel role for this highly conserved threonine (Thr303) in the function of P450 2E1. We found that the P450 2E1 T303A mutant undergoes rapid autoinactivation in the reconstituted system during catalytic turnover when the electrons are provided by NADPH. This inactivation was much faster than that of the wild type P450 2E1 and was prevented by catalase. Both the P450 2E1 wild type and T303A mutants produce hydrogen peroxide during the incubations. The inactivation was accompanied by heme destruction with part of the heme becoming covalently attached to protein. The heme destruction was prevented by catalase or by the presence of substrate. Interestingly, this inactivation occurred much more rapidly in the presence of both an electron transfer system and hydrogen peroxide externally added to the enzyme. This accelerated inactivation during catalytic turnover was also found with a 2B4 T302A mutant, which corresponds to 2E1 T303A. Our results suggest that the conserved threonine in these P450s prevents rapid autoinactivation during the catalytic cycle and that this residue may be highly conserved in P450s since it allows them to remain catalytically active for longer periods of time.
Asunto(s)
Citocromo P-450 CYP2E1/química , Peróxido de Hidrógeno/farmacología , Antioxidantes/química , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2E1/genética , Inhibidores del Citocromo P-450 CYP2E1 , Transporte de Electrón , Electroforesis en Gel de Poliacrilamida , Hemo/química , Mutación , NADP/químicaRESUMEN
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
Phencyclidine (PCP) is a mechanism-based inactivator of cytochrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphetamine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-octylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k(1) = 5.0 s(-1), A(1) = 30%; k(2) = 0.02 s(-1), A(2) = 70%, where A(2) indicates the fractional magnitude of the second phase) compared with the native enzyme (k(1) = 8.0 s(-1), A(1) = 58%; k(2) = 0.3 s(-1), A(2) = 42%). Analysis of benzphetamine metabolism by the inactivated protein using liquid chromatography/electrospray ionization/mass spectrometry showed that the rates of formation of nor-benzphetamine and hydroxylated nor-benzphetamine were decreased by 75 and 69%, respectively, whereas the rates of formation for amphetamine, hydroxybenzphetamine, and methamphetamine showed slight but statistically insignificant decreases after the inactivation. The rate of reduction of P450 2B6 by NADPH and reductase was decreased by 6-fold as a result of the modification by PCP. In addition, the extent of uncoupling of NADPH oxidation from product formation, a process leading to futile production of H(2)O(2), increased significantly during the metabolism of ethylbenzene as a result of the inactivation.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Fenciclidina/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Benzfetamina/farmacocinética , Catálisis , Cromatografía Liquida , Citocromo P-450 CYP2B6 , Transporte de Electrón , Oxidorreductasas N-Desmetilantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por SustratoRESUMEN
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
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
The P450 type cytochromes are responsible for the metabolism of a wide variety of xenobiotics and endogenous compounds. Although P450-catalyzed reactions are generally thought to lead to detoxication of xenobiotics, the reactions can also produce reactive intermediates that can react with cellular macromolecules leading to toxicity or that can react with the P450s that form them leading to irreversible (i.e., mechanism-based) inactivation. This perspective describes the fundamentals of mechanism-based inactivation as it pertains to P450 enzymes. The experimental approaches used to characterize mechanism-based inactivators are discussed, and the criteria required for a compound to be classified as a mechanism-based inactivator are outlined. The kinetic scheme for mechanism-based inactivation and the calculation of the relevant kinetic constants that describe a particular inactivation event are presented. The structural aspects and important functional groups of several classes of molecules that have been found to impart mechanism-based inactivation upon metabolism by P450s such as acetylenes, thiol-containing compounds that include isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, and cyclic tertiary amines are described. Emphasis throughout this perspective is placed on more recent findings with human P450s where the site of modification, whether it be the apoprotein or the heme moiety, and, at least in part, the identity of the reactive intermediate responsible for the loss in P450 activity are known or inferred. Recent advances in trapping procedures as well as new methods for identification of reactive intermediates are presented. A variety of clinically important drugs that act as mechanism-based inactivators of P450s are discussed. The irreversible inactivation of human P450s by these drugs has the potential for causing serious drug-drug interactions that may have severe toxicological effects. The clinical significance of inactivating human P450s for improving drug efficacy as well as drug safety is discussed along with the potential for exploiting mechanism-based inactivators of P450s for therapeutic benefits.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Aminoácidos/química , Animales , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Humanos , Cinética , Relación Estructura-Actividad , Xenobióticos/metabolismoRESUMEN
The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single mono-oxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent Km of 0.7 microM. The apparent Km values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 microM, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Sistema Enzimático del Citocromo P-450/fisiología , Riñón/metabolismo , Microsomas Hepáticos/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Citocromo P-450 CYP3A , Endocannabinoides , Compuestos Epoxi/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ácidos Hidroxieicosatetraenoicos/metabolismo , Cinética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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
Cytochromes P450 (P450) 2B6 and 3A5 are inactivated by bergamottin (BG). P450 2B6 metabolized BG primarily to M3 and M4 and one minor metabolite (M1). The metabolites were analyzed, and the data indicated that M1 was bergaptol, M3 was 5'-OH-BG, and M4 was a mixture of 6'- and 7'-OH-BG. Because 6'- and 7'-OH-BG were the primary metabolites, it suggested that P450 2B6 preferentially oxidized the geranyloxy chain of BG. Metabolism of BG by P450 3A5 resulted in three major metabolites: [bergaptol, M3 (5'-OH-BG), and M5 (2'-OH-BG)], and two minor metabolites [M2 (6',7'-dihydroxy-BG) and M4 (6'- and 7'-OH-BG)]. Because bergaptol was the most abundant metabolite formed, it suggested that P450 3A5 metabolized BG mainly by cleaving the geranyl-oxy chain. Molecular modeling studies confirmed that docking of BG in the P450 2B6 active site favors oxidation in the terminal region of the geranyl-oxy chain, whereas positioning the 2'-carbon of BG nearest the heme iron is preferred by P450 3A5. Glutathione (GSH)-BG conjugates were formed by both P450. Each enzyme predominantly formed conjugates with m/z values of 662. Tandem mass spectrometry analysis of the GSH conjugates indicated that the oxidation forming a reactive intermediate occurred on the furan moiety of BG, presumably through the initial formation of an epoxide at the furan double bond. The data indicate that oxidation of the geranyl-oxy chain resulted in the formation of stable metabolites of BG, whereas oxidation of the furan ring produced reactive intermediates that may be responsible for binding to and inactivating P450 2B6 and 3A4.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/fisiología , Sistema Enzimático del Citocromo P-450/fisiología , Furocumarinas/metabolismo , Oxidorreductasas N-Desmetilantes/fisiología , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B6 , Furocumarinas/química , Glutatión/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de MasasRESUMEN
Efavirenz is a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor used in combination therapy to treat HIV-1. Efavirenz metabolism is catalyzed primarily by the polymorphic enzyme P450 2B6. Metabolism of efavirenz by P450 2B6 and the naturally occurring P450 2B6.4 mutant led to the formation of 8-hydroxyefavirenz. Efavirenz inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin activity of the wild-type P450 2B6 enzyme in a time-, concentration-, and NADPH-dependent manner. However, the P450 2B6.4 variant was not inactivated by efavirenz. The ability of efavirenz to inactivate both enzymes was investigated using cyclophosphamide and bupropion, two structurally unrelated substrates of P450 2B6, as probes. Preincubations with efavirenz decreased the ability of the wild-type enzyme to hydroxylate both substrates to similar extents but had no effect on the activities of the mutant enzyme. Interestingly, the inactivation of the wild-type enzyme was completely reversible after 24 h of dialysis as determined by heme, reduced CO spectra, and activity loss. In contrast, 8-hydroxyefavirenz, a metabolite of efavirenz, was able to inactivate both enzymes irreversibly. These data suggest that incubations of P450 2B6 and P450 2B6.4 with either the parent compound efavirenz or the metabolite 8-hydroxyefavirenz in the reconstituted system result in the formation of two different reactive intermediates that lead to losses in enzymatic activity by two different mechanisms, one reversible and one irreversible.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Oxazinas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Alquinos , Sustitución de Aminoácidos/genética , Hidrocarburo de Aril Hidroxilasas/genética , Benzoxazinas , Ciclopropanos , Citocromo P-450 CYP2B6 , Activación Enzimática/genética , Humanos , Oxazinas/química , Oxidorreductasas N-Desmetilantes/genéticaRESUMEN
The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Etinilestradiol/análogos & derivados , Etinilestradiol/farmacología , Glutatión/química , Péptidos/efectos de los fármacos , Animales , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Etinilestradiol/química , Glutatión/metabolismo , Humanos , Estructura Molecular , Péptidos/metabolismo , Ratas , Ratas Long-Evans , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Relación Estructura-ActividadRESUMEN
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
Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K(I), 5 microM; k(inact) 0.09 min(-1); and t1/2, 8 min. The kinetic constants obtained for the inactivation of the testosterone 6beta-hydroxylation activity of P450 3A5 were: K(I), 20 microM; k(inact) 0.045 min(-1); and t1/2, 15 min. Incubations of P450s 2B6 and 3A5 with 20 microM BG at 37 degrees C for 20 min resulted in an approximately 60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were approximately 2 and approximately 20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [14C]BG was irreversibly bound to the apoprotein in the BG-inactivated samples. The stoichiometry of binding was approximately 0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6',7'-dihydroxybergamottin and bergaptol.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Bebidas , Citrus , Inhibidores Enzimáticos del Citocromo P-450 , Furocumarinas/farmacología , Hemo/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Monóxido de Carbono/metabolismo , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Electroforesis en Gel de Poliacrilamida , Furocumarinas/metabolismo , Técnicas In Vitro , Cinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Oxidorreductasas N-Desmetilantes/metabolismo , Unión Proteica , Ratas , Ratas Long-Evans , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds.
Asunto(s)
Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/metabolismo , Hierro/metabolismo , Treonina/metabolismo , Alquinos/farmacología , Anaerobiosis , Animales , Derivados del Benceno/farmacología , Sitios de Unión , Catálisis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2E1/genética , Inhibidores del Citocromo P-450 CYP2E1 , Activación Enzimática/efectos de los fármacos , Hierro/química , Modelos Moleculares , Estructura Molecular , Mutación/genética , Estructura Terciaria de Proteína , Conejos , Análisis Espectral , Treonina/genética , terc-Butilhidroperóxido/farmacologíaRESUMEN
We have previously demonstrated that substituting Val for Thr-205 in P450 2B1 abolishes the 16beta-hydroxylation of testosterone and markedly decreases the ability of 2-ethnylnaphthalene (2EN) and 17alpha-ethynylestradiol (17EE) to inactivate P450 2B1. The role of Thr-205 has been further investigated by measuring the kinetics of the mechanism-based inactivation of the 7-ethoxy-(trifluoromethyl)coumarin deethylation activity of 2B1 by 2EN and 17EE in wild-type (WT) and mutant P450s. In general, the kinetics of the inactivation of the Ser and Ala mutants was not significantly altered compared with WT. In contrast, the efficiency of the inactivation of the Val mutant decreased by approximately 6- and approximately 30-fold for 2EN and 17EE, respectively. High-pressure liquid chromatography (HPLC) analysis and SDS gel electrophoresis demonstrated the covalent binding of radiolabeled 2EN- and 17EE-reactive intermediates to the WT apoprotein, but not the Val mutant. The Val mutant was able to metabolize 2EN to 2-naphthylacetic acid, except the initial rate was slower than the WT. HPLC analysis of the 17EE incubation mixtures revealed three major metabolites and showed a correlation between the efficiency of inactivation and the generation of one of the major metabolites (C). Metabolite C was generated by the WT, Ser mutant, and Ala mutant. Metabolite C may be formed by the oxidation of the ethynyl group, and this reactive intermediate contributes to the inactivation of P450 2B1 by 17EE. The site-specific mutation of one residue, Thr-205 to Val, is sufficient to alter the profile of products formed during 17EE metabolism, such that very low levels of metabolite C are formed and inactivation is essentially abolished.
Asunto(s)
Citocromo P-450 CYP2B1/antagonistas & inhibidores , Etinilestradiol/farmacología , Naftalenos/farmacología , Treonina/fisiología , Sustitución de Aminoácidos , Apoproteínas/metabolismo , Autorradiografía , Sitios de Unión , Catálisis , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Etinilestradiol/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Cinética , Modelos Moleculares , Naftalenos/metabolismo , Conformación Proteica , ValinaRESUMEN
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
The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Inhibidores Enzimáticos/metabolismo , Isoenzimas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Tiotepa/metabolismo , Aminas/metabolismo , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Benzfetamina/metabolismo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B6 , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Testosterona/metabolismo , Tiotepa/farmacologíaRESUMEN
Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity. Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from benzphetamine was much less affected. Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of benzphetamine. Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.
Asunto(s)
Benzfetamina/metabolismo , Cumarinas/metabolismo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/metabolismo , Inhibidores Enzimáticos/farmacología , Testosterona/metabolismo , Triazoles/farmacología , Anaerobiosis , Animales , Benzfetamina/química , Cumarinas/química , Citocromo P-450 CYP2B1/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Peróxido de Hidrógeno/metabolismo , Hidroxilación , Masculino , Microsomas Hepáticos/enzimología , NADP/metabolismo , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Unión Proteica , Ratas , Ratas Long-Evans , Testosterona/química , Triazoles/química , Triazoles/metabolismoRESUMEN
This report investigates and characterizes the mechanism for the novel reversible inactivation of a T303A mutant of rabbit cytochrome P450 (P450) 2E1 by tert-butyl acetylene (tBA). P450 2E1 T303A was inactivated in a time-, concentration-, and NADPH-dependent manner through the formation of two tBA adducts to the P450 heme. Interestingly, losses in enzymatic activity and in the reduced CO spectrum of the tBA-inactivated T303A mutant could be restored to the samples after an overnight incubation at 4 degrees C. Removal of free tBA and NADPH from the tBA-inactivated T303A samples by spin column gel filtration demonstrated that the observed reversibility was time-dependent and was not significantly affected by the presence or absence of NADPH or tBA. Furthermore, the recovery of native heme was dependent on the native P450 enzyme structure. Electrospray ionization liquid chromatography-tandem mass spectrometry analysis under nondenaturing conditions of a preacidified tBA-inactivated T303A sample yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns characteristic of a tBA adduct to the P450 heme. These adducts were absent in nonacidified samples subjected to the same conditions. In contrast, tandem mass spectrometry analysis of both non- and preacidified tBA-inactivated wild-type 2E1 samples yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns similar to the preacidified T303A mutant adducts. These results lend insight into the reversible inactivation mechanism of the tBA-inactivated T303A mutant and suggest a role for the highly conserved threonine 303 residue in proton donation to the P450 2E1 active site and the stabilization of a reactive intermediate during substrate metabolism by P450.