RESUMEN
17α-Ethynylestradiol (EE), a major component of many oral contraceptives, affects the activities of a number of the human cytochrome P450 (P450) enzymes. Here, we characterized the effect of EE on CYP2J2, a major human P450 isoform that participates in metabolism of arachidonic acid. EE inactivated the hydroxyebastine carboxylation activity of CYP2J2 in a reconstituted system. The loss of activity is time and concentration dependent and requires NADPH. The KI and kinact values for the inactivation were 3.6 µM and 0.08 minute-1, respectively. Inactivation of CYP2J2 by EE was due to formation of a heme adduct as well as an apoprotein adduct. Mass spectral analysis of CYP2J2 partially inactivated by EE showed two distinct protein masses in the deconvoluted spectrum that exhibited a mass difference of approximately 312 Da, which is equivalent to the sum of the mass of EE and one oxygen atom. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a heme adduct with MH+ ion at m/z 875.5, corresponding to alkylation of an iron-depleted prosthetic heme by EE plus one oxygen atom. The reactive intermediate responsible for covalently modifying both the prosthetic heme and apoprotein was characterized by trapping with glutathione (GSH). LC-MS/MS analysis revealed two GSH conjugate isomers with MH+ ions at m/z 620, which were formed by reaction between GSH and EE with the oxygen being added to either the internal or terminal carbon of the ethynyl moiety. High-pressure liquid chromatography analysis revealed that three other major metabolites were formed during EE metabolism by CYP2J2.
Asunto(s)
Apoproteínas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Etinilestradiol/farmacología , Hemo/metabolismo , Ácido Araquidónico/metabolismo , Citocromo P-450 CYP2J2 , Glutatión/metabolismo , Humanos , NADP/metabolismo , Oxígeno/metabolismoRESUMEN
The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: N-(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: KI of 6.1 µM and kinact of 0.22 min-1 for MS and KI of 2.5 µM and kinact of 0.05 min-1 for OD. The partition ratios for MS and OD were â¼10 and â¼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a Ks of 2.8 µM and the IC50 for loss of OHEB carboxylation activity was 0.18 µM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.
Asunto(s)
Alquinos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/metabolismo , Alquinos/química , Alquinos/metabolismo , Amidas/química , Amidas/metabolismo , Amidas/farmacología , Butirofenonas/metabolismo , Cromatografía Liquida , Citocromo P-450 CYP2J2 , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/química , Danazol/química , Danazol/metabolismo , Danazol/farmacología , Activación Enzimática/efectos de los fármacos , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Hemo/química , Humanos , Cinética , Piperidinas/metabolismo , Espectrometría de Masas en TándemRESUMEN
The crystal structures of human CYP2B6 indicate that Phe206 and Val367 are in close proximity to the substrate binding site and suggest that both residues may play important roles in substrate metabolism and inhibitor binding. To test this hypothesis, we investigated the effects of mutating these residues to Ala on the regiospecificity of CYP2B6 for the metabolism of testosterone and androstenedione. For testosterone metabolism, 16ß-OH-testosterone formation by the F206A mutant was <5% of the wild type (WT), whereas the V367A mutant exhibited a doubling of 16α-OH-testosterone formation with a 50% decrease in 16ß-OH-testosterone formation compared with the WT. Significant alterations in the regiospecificity for androstenedione metabolism were also observed. To investigate the roles of these two residues in the metabolic activation of mechanism-based inactivators, tert-butylphenylacetylene (BPA) and bergamottin (BG) were used to test the susceptibility to inactivation. Although the rates of inactivation of both mutants by BG were not significantly decreased compared with the WT, the efficiency of inactivation by BPA of both mutants was more than an order of magnitude lower. Our results demonstrate that Phe206 plays a crucial role in determining the specificity of CYP2B6 for the 16ß-hydroxylation of testosterone and androstenedione and that it also plays an important role in BG binding and mechanism-based inactivation by BPA. In addition, Val367 dramatically enhances the catalytic activity of CYP2B6 toward androstenedione and plays an important role in mechanism-based inactivation by BPA. The results presented here show the important roles of Phe206 and Val367 in interactions of CYP2B6 with substrates and inactivators/inhibitors and are consistent with the crystal structures.
Asunto(s)
Andrógenos/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Androstenodiona/metabolismo , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Inhibidores Enzimáticos/farmacología , Humanos , Hidroxilación , Cinética , Mutación , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/genética , Testosterona/metabolismoRESUMEN
The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a K(I) of 0.9 µM, a k(inact) of 0.05 min⻹, and a partition ratio of approximately 3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the protonated molecular ion of RTV exhibits an m/z at 721 and its two major metabolites are an oxidation product with MH⺠at m/z 737 and a deacylated product with MH⺠at m/z 580. Inactivation of CYP2B6 by incubation with 10 µM RTV for 10 min resulted in an approximately 50% loss of catalytic activity and native heme, but no modification of the apoprotein was observed. RTV was found to be a potent mixed-type reversible inhibitor (K(i) = 0.33 µM) and a type II ligand (spectral dissociation constant-K(s) = 0.85 µM) of CYP2B6. Although previous studies have demonstrated that RTV is a potent mechanism-based inactivator of CYP3A4, the molecular mechanism responsible for the inactivation has not been determined. Here, we provide evidence that RTV inactivation of CYP3A4 is due to heme destruction with the formation of a heme-protein adduct. Similar to CYP2B6, there is no significant modification of the apoprotein. Furthermore, LC-MS/MS analysis revealed that both CYP3A4 and human liver microsomes form an RTV-glutathione conjugate having a MH⺠at m/z 858 during metabolism of RTV, suggesting the formation of an isocyanate intermediate leading to formation of the conjugate.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Catálisis/efectos de los fármacos , Inhibidores del Citocromo P-450 CYP3A , Hemo/metabolismo , Ritonavir/farmacología , Apoproteínas/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/metabolismo , Glutatión/metabolismo , Humanos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismoRESUMEN
Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent K(M) and k(cat) of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR increased up to 23-fold with virtually no change in the k(cat) for the reaction, however, at higher concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the k(cat) also decreased by 11-fold. Additionally, CYP2E1 increased the apparent K(M) of CYP2B4 for BNZ by 8-fold and the apparent K(M) did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent K(M) values of CYP2B4 and CYP2E1 for CPR are similar, the apparent K(M) of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Secuencia de Bases , Catálisis , Familia 2 del Citocromo P450 , Cartilla de ADN , Hidroxilación , Especificidad por SustratoRESUMEN
Previous studies have demonstrated that bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of CYP3A4 and contributes, in part, to the grapefruit juice-drug interaction. Although the covalent binding of [(14)C]BG to the CYP3A4 apoprotein has been demonstrated by SDS-polyacrylamide gel electrophoresis, the identity of the modified amino acid residue and the reactive intermediate species of BG responsible for the inactivation have not been reported. In the present study, we show that inactivation of CYP3A4 by BG results in formation of a modified apoprotein-3A4 and a GSH conjugate, both exhibiting mass increases of 388 Da, which corresponds to the mass of 6',7'-dihydroxybergamottin (DHBG), a metabolite of BG, plus one oxygen atom. To identify the adducted residue, BG-inactivated 3A4 was digested with trypsin, and the digests were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). A mass shift of 388 Da was used for the SEQUEST database search, which revealed a mass increase of 388 Da for the peptide with the sequence (272)LQLMIDSQNSK(282), and MS/MS analysis of the adducted peptide demonstrated that Gln273 is the residue modified. Mutagenesis studies showed that the Gln273 to Val mutant was resistant to inactivation by BG and DHBG and did not generate two of the major metabolites of BG formed by 3A4 wild type. In conclusion, we have determined that the reactive intermediate, oxygenated DHBG, covalently binds to Gln273 and thereby contributes to the mechanism-based inactivation of CYP3A4 by BG.
Asunto(s)
Bebidas , Citrus paradisi , Inhibidores del Citocromo P-450 CYP3A , Interacciones Alimento-Droga , Furocumarinas/metabolismo , Furocumarinas/farmacología , Sustitución de Aminoácidos , Apoenzimas/metabolismo , Sitios de Unión , Cromatografía Liquida , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Glutatión/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Plásmidos , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , Citocromo P-450 CYP3A/química , Ditionita/metabolismo , Humanos , Oxidación-Reducción , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Tirosina/metabolismoRESUMEN
A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.
Asunto(s)
Acetileno/análogos & derivados , Hidrocarburo de Aril Hidroxilasas/química , Acetileno/química , Acetileno/metabolismo , Secuencia de Aminoácidos , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Sitios de Unión , Dominio Catalítico , Biología Computacional , Cristalografía por Rayos X , Familia 2 del Citocromo P450 , Medición de Intercambio de Deuterio , Dimerización , Modelos Moleculares , Conformación Proteica , ConejosRESUMEN
An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b(5) and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b(5). The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a K(I) of 24 µM and a k(inact) of 0.04 min(-1). This K(I) is significantly greater than the clinical OSI-930 C(max) of 1.7 µM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation of the thiophene sulfur to give the sulfoxide, which has previously been shown to be a significant metabolite of OSI-930. Because OSI-930 is an inactivator of P450 3A4 but does not exhibit any effect on P450 3A5 activity under the same conditions, it may be an appropriate probe for exploring unique aspects of these two very similar P450s.
Asunto(s)
Antineoplásicos/metabolismo , Inhibidores del Citocromo P-450 CYP3A , Quinolinas/metabolismo , Tiofenos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Sitios de Unión , Biotransformación , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A/genética , Citocromos b5/farmacología , Citocromos b5/fisiología , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Humanos , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Quinolinas/farmacocinética , Quinolinas/farmacología , Tiofenos/farmacocinética , Tiofenos/farmacologíaRESUMEN
The mechanism-based inactivation of human CYP2B6 by tert-butylphenylacetylene (BPA) in the reconstituted system was investigated. The inactivation of CYP2B6 by BPA is time-, concentration-, and NADPH-dependent and exhibits a K(I) of 2.8 µM, a k(inact) of 0.7 min(-1), and a t(1/2) of 1 min. The partition ratio is â¼5. Unlike CYP2B1 and CYP2B4, in addition to the formation of an apoprotein adduct and a glutathione conjugate, a small heme adduct was observed when CYP2B6 was incubated with BPA. The mass increase of the adducted apoprotein and GSH conjugate is 174 Da, equivalent to the mass of one molecule of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B6 was digested with trypsin, and the digest was then analyzed by liquid chromatography-tandem mass spectrometry. A mass shift of 174 Da was used for the SEQUEST database search, and the identity of the modified residue was confirmed by MS/MS fragmentation of the modified peptide. Two residues, Lys274 and Thr302, were identified as having been modified. Further mutagenesis studies have demonstrated that the residue that is modified to result in inactivation is Thr302, not Lys274. Docking studies show that in the enzyme-substrate complex, Thr302 is in close contact with the triple bond of BPA with a distance of 3.8 Å between the terminal carbon of BPA and the oxygen in the hydroxyl group of Thr302. In conclusion, Thr302 of CYP2B6 is covalently modified by a reactive metabolite of BPA, and this modification is responsible for the mechanism-based inactivation.
Asunto(s)
Acetileno/análogos & derivados , Hidrocarburo de Aril Hidroxilasas/metabolismo , Inhibidores Enzimáticos/farmacología , Oxidorreductasas N-Desmetilantes/metabolismo , Acetileno/farmacología , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Cromatografía Liquida , Citocromo P-450 CYP2B6 , Cartilla de ADN , Humanos , Mutagénesis Sitio-Dirigida , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/genética , Espectrometría de Masas en TándemRESUMEN
Cytochromes P450 (CYPs or P450s) contain a highly conserved threonine residue in the active site, which is referred to as Thr302 in the amino acid sequence of CYP2B4. Extensive biochemical and crystallographic studies have established that this Thr302 plays a critical role in activating molecular oxygen to generate Compound I, a putative iron(IV)-oxo porphyrin cation radical, that carries out the preliminary oxygenation of CYP substrates. Because of its proximity to the center of the P450 active site, this Thr302 is susceptible to mechanism-based inactivation under certain conditions. In this article, we review recent studies on the mechanism-based inactivation of three mammalian P450s in the 2B family, CYP2B1 (rat), 2B4 (rabbit) and 2B6 (human) by tert-butylphenylacetylene (tBPA). These studies showed that tBPA is a potent mechanism-based inactivator of CYP2B1, 2B4 and 2B6 with high k(inact)/K(I) ratios (0.23-2.3min(-1)µM(-1)) and low partition ratios (0-5). Furthermore, mechanistic studies revealed that tBPA inactivates these three CYP2B enzymes through the formation of a single ester adduct with the Thr302 in the active site. These inhibitory properties of tBPA allowed the preparation of a modified CYP2B4 where the Thr302 was covalently and stoichiometrically labeled by a reactive intermediate of tBPA in quantities large enough to permit spectroscopic and crystallographic studies of the consequences of covalent modification of Thr302. Molecular modeling studies revealed a unique binding mode of tBPA in the active site that may shed light on the potency of this inhibition. The results from these studies may serve as a basis for designing more specific and potent inhibitors for P450s by targeting this highly conserved threonine residue which is present in the active sites of most mammalian P450s.
Asunto(s)
Acetileno/análogos & derivados , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Treonina/metabolismo , Acetileno/química , Acetileno/metabolismo , Secuencia de Aminoácidos , Animales , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Especificidad por Sustrato , Treonina/químicaRESUMEN
The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence (296)FFAGTSSTTLR(308) in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 A, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.
Asunto(s)
Acetileno/análogos & derivados , Citocromo P-450 CYP2B1/efectos de los fármacos , Treonina/efectos de los fármacos , Acetileno/farmacología , Secuencia de Aminoácidos , Electrocromatografía Capilar , Dominio Catalítico/efectos de los fármacos , Citocromo P-450 CYP2B1/química , Bases de Datos de Proteínas , Escherichia coli , Hemo/química , Hemo/metabolismo , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Análisis Espectral , Treonina/química , TripsinaRESUMEN
Although the ability of disulfiram to inactivate CYP2E1 has been known for more than 20 years, the mechanism has not yet been elucidated. A metabolite of disulfiram, diethyldithocarbamate (DDC), is converted by CYP2E1 to a reactive intermediate that subsequently inactivates the protein, leading to mechanism-based inactivation. Mass spectral analysis of the inactivated human 2E1 protein demonstrates that the inactivation is due to the formation of an adduct of the reactive metabolite of DDC with the apoprotein. These data, along with mass spectral analysis of a reactive intermediate trapped with GSH, indicate the involvement of a reactive intermediate with a molecular mass of 116 Da. Our results suggest that this binding involves formation of a disulfide bond with one of the eight cysteines in CYP2E1. The inactivation of wild-type CYP2E1 as well as two of its polymorphic mutants, CYP2E1*2 and CYP2E1*4, was also investigated. For wild-type CYP2E1, the K(I) was 12.2 µM and the k(inact) was 0.02 min(-1). The K(I) values for the two polymorphic mutants were 227.6 and 12.4 µM for CYP2E1.2 and CYP2E1.4, and the k(inact) values were 0.0061 and 0.0187 min(-1), respectively. These data indicate that DDC is a much less efficient inactivator of CYP2E1.2 than it is of either the wild-type or the CYP2E1.4 variant.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2E1 , Ditiocarba/farmacología , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2E1/genética , Disulfiram/farmacología , Ditiocarba/metabolismo , Humanos , Cinética , Mutación , Espectrometría de Masas en TándemRESUMEN
We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
Asunto(s)
Acetileno/análogos & derivados , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Acetileno/farmacología , Benzfetamina/farmacología , Catálisis/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Familia 2 del Citocromo P450 , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estereoisomerismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Mifepristone [RU486; 17beta-hydroxy-11beta-(4-dimethylaminophenyl)-17alpha-(1-propynyl)-estra-4,9-dien-3-one] inactivates CYP2B6 in the reconstituted system in a mechanism-based manner. The loss of 7-ethoxy-4-(trifluoromethyl)-coumarin deethylation activity of CYP2B6 is concentration- and time-dependent. The inactivation requires NADPH and is irreversible. The concentration of inactivator required to give the half-maximal rate of inactivation is 2.8 microM, and the maximal rate constant for inactivation at a saturating concentration of the inactivator is 0.07 min(-1). Incubation of CYP2B6 with 20 microM RU486 for 15 min resulted in 61% loss of catalytic activity, 60% loss of the reduced cytochrome P450 (P450)-CO complex, and a 40% loss of native heme. The partition ratio is approximately 5, and the stoichiometry of binding is approximately 0.6 mol RU486/mol P450 inactivated. SDS-polyacrylamide gel electrophoresis and high-pressure liquid chromatography analysis showed that [(3)H]RU486 was irreversibly bound to CYP2B6 apoprotein. RU486 is metabolized to form three major metabolites and bioactivated to give reactive intermediates by purified P450s in the reconstituted system. After incubation of RU486 with the purified P450s and liver microsomes from rats and humans in the presence of glutathione (GSH) and NADPH, GSH conjugates with MH(+) ions at m/z 769, 753, and 751 were detected by liquid chromatography-tandem mass spectrometry. Two GSH conjugates with MH(+) ions at m/z 753 are formed from the reaction of GSH with RU486. The adducts are formed after addition of an activated oxygen to the carbon-carbon triple bond of the propynyl moiety. This suggests that oxirene intermediates may be involved in the mechanism of inactivation. It seems that the potential for drug-drug interactions of RU486 may not be limited only to CYP3A4 and should also be evaluated for drugs metabolized primarily by CYP2B6, such as bupropion and efavirenz.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Antagonistas de Hormonas/metabolismo , Mifepristona/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Apoproteínas/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Cristalización , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/biosíntesis , Dexametasona/farmacología , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática/efectos de los fármacos , Glutatión/metabolismo , Hemo/antagonistas & inhibidores , Hemo/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas/biosíntesis , Isoenzimas/metabolismo , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Fenobarbital/farmacología , Ratas , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.
Asunto(s)
Acetileno/análogos & derivados , Alquinos/farmacología , Apoproteínas/metabolismo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/genética , Inhibidores Enzimáticos/farmacología , Éteres/farmacología , Hemo/metabolismo , Mutación/efectos de los fármacos , Acetileno/farmacología , Sustitución de Aminoácidos , Apoproteínas/química , Apoproteínas/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B1/química , Glutatión/metabolismo , Hemo/química , Humanos , Cinética , Espectrometría de Masas , Modelos Moleculares , Oxígeno/química , Conformación Proteica/efectos de los fármacos , Solubilidad , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
17Alpha-ethynylestradiol (EE) inactivates cytochrome P450 3A5 (3A5) in the reconstituted system in a mechanism-based manner. The inactivation is dependent on NADPH, and it is irreversible. The inactivation of 3A5 by EE is also dependent on cytochrome b5 (b5). The values for the K(I) and k(inact) of the 7-benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation activity of 3A5 are 26 microM and 0.06 min(-1), respectively. Incubation of 3A5 with EE resulted in a 62% loss of catalytic activity, 60% loss in the reduced CO difference spectrum, and 40% decrease in native heme with the formation of a heme adduct. The partition ratio was approximately 25, and the stoichiometry of binding was approximately 0.3 mol of EE metabolite bound/mol of P450 inactivated. Four major metabolites were formed during the metabolism of EE by 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [3H]EE was irreversibly bound to 3A5 apoprotein. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) revealed that two glutathione (GSH) conjugates with m/z values of 620 were formed only in the presence of b5. These two conjugates are formed from the reaction of GSH with the ethynyl group with the oxygen being inserted into either the internal or terminal carbon. A heme adduct with the ion at m/z 927 and two dipyrrole adducts with ions at m/z 579 were detected by LC-MS/MS analysis. In conclusion, 3A5 can activate EE to a 17alpha-oxirene-related reactive species that can then partition the oxygen between the internal and terminal carbons of the ethynyl group to form heme and apoprotein adducts, resulting in the inactivation of P450 3A5.
Asunto(s)
Apoproteínas/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Citocromos b5/fisiología , Inhibidores Enzimáticos/farmacología , Etinilestradiol/farmacología , Glutatión/metabolismo , Hemo/metabolismo , Biotransformación , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Citocromo P-450 CYP3A , Endopeptidasas/química , Humanos , Espectrometría de Masas , NADP/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
This study examined the reaction of peroxynitrite (PN) with two human cytochrome P450s, P450 2B6 (2B6) and P450 2E1 (2E1). After the reaction with PN, the NADPH/reductase-supported 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) deethylation activity of both P450s was decreased in a concentration-dependent manner. HPLC analysis revealed that the prosthetic heme group of 2B6 was modified but to a lesser extent than the decrease in enzymatic activity. In contrast, the heme moiety of 2E1 was not altered. These results suggest that protein modification by PN contributed to the loss in enzymatic activity of 2B6 and 2E1 but to different extents. After trypsin digestion of the control and PN-inactivated P450s, tyrosine nitration was used as a biomarker for protein modification and the addition of the nitro group was determined using electrospray ionization-liquid chromatography-tandem mass spectrometry, allowing site-specific assignment of the tyrosine residues nitrated. Tyrosine residues 354, 244, 268, and 380 in 2B6 and tyrosine residues 317, 422, 69, and 380 in 2E1 were found to be nitrated. Tyrosine 354 is the primary site of nitration in 2B6, and tyrosine residues 422 and 317 are the primary targets for nitration in 2E1. After PN exposure, the EFC catalytic activity of 2E1 supported by tert-butylhydroperoxide was not affected, and the activity of 2B6 supported by tert-butylhydroperoxide was decreased to a lesser extent than that supported by NADPH/reductase. Following exposure to PN, the levels of the reduced-CO complex were less than the content of native heme remaining. These results suggest that PN-mediated protein modification has no effect on substrate binding but may impair the interaction of the reductase with P450s, thereby inhibiting electron transfer. Homology modeling shows that Tyr422 of 2E1 is in close proximity to the FMN domain of reductase, suggesting that Tyr422 may be involved in transferring electrons from the reductase to the heme and thus may play a critical structural and functional role in the extensive activity loss following PN exposure.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores del Citocromo P-450 CYP2E1 , Inhibidores Enzimáticos/farmacología , Hemo/química , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Ácido Peroxinitroso/farmacología , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Monóxido de Carbono/química , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Tirosina/biosíntesis , terc-Butilhidroperóxido/farmacologíaRESUMEN
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
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.