RESUMO
The cytochrome P450 enzyme CYP102A1 from Bacillus megaterium is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was â¼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredutases/metabolismo , Multimerização Proteica , Proteínas de Bactérias/química , Catálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/química , Transporte de Elétrons , Heme/química , Modelos Moleculares , Simulação de Dinâmica Molecular , NADPH-Ferri-Hemoproteína Redutase/química , Oxirredutases/química , Conformação ProteicaRESUMO
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.
Assuntos
Apoproteínas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Etinilestradiol/farmacologia , Heme/metabolismo , Ácido Araquidônico/metabolismo , Citocromo P-450 CYP2J2 , Glutationa/metabolismo , Humanos , NADP/metabolismo , Oxigênio/metabolismoRESUMO
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.
Assuntos
Alcinos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , Alcinos/química , Alcinos/metabolismo , Amidas/química , Amidas/metabolismo , Amidas/farmacologia , Butirofenonas/metabolismo , Cromatografia Líquida , Citocromo P-450 CYP2J2 , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/química , Danazol/química , Danazol/metabolismo , Danazol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Heme/química , Humanos , Cinética , Piperidinas/metabolismo , Espectrometria de Massas em TandemRESUMO
According to the Centers for Disease Control and Prevention, the incidence of inflammatory bowel diseases (IBD) is about 1 in 250 people in the United States. The disease is characterized by chronic or recurring inflammation of the gut. Because of the localization of the endocannabinoid system in the gastrointestinal tract, it may be a potential pharmacologic target for the treatment of IBD and other diseases. Fatty acid amide hydrolase (FAAH) is a potential candidate because it is upregulated in IBD. FAAH hydrolyzes and, as a consequence, inactivates anandamide (AEA), a prominent endocannabinoid. Inhibition of FAAH would lead to increases in the amount of AEA oxidized by cytochrome P450s (P450s). CYP2J2, the major P450 epoxygenase expressed in the heart, is also expressed in the intestine and has previously been reported to oxidize AEA. We have investigated the possibility that it may play a role in AEA metabolism in the gut and have demonstrated that purified human CYP2J2 metabolizes AEA to form the 20-hydroxyeicosatetraenoic acid ethanolamide (HETE-EA) and several epoxygenated products, including the 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs), in the reconstituted system. Kinetic studies suggest that the KM values for these products range from approximately 10 to 468 µM and the kcat values from 0.2 to 23.3 pmol/min per picomole of P450. Human intestinal microsomes, which express CYP2J2, metabolize AEA to give the 5,6-, 8,9-, and 11,12-EET-EAs, as well as 20-HETE-EA. Studies using specific P450 inhibitors suggest that although CYP2J2 metabolizes AEA, it is not the primary P450 responsible for AEA metabolism in human intestines.
Assuntos
Ácidos Araquidônicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Endocanabinoides/metabolismo , Intestinos/citologia , Microssomos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ácido Araquidônico/farmacologia , Citocromo P-450 CYP2J2 , Humanos , Proteínas Recombinantes/metabolismoRESUMO
Clopidogrel is a prodrug that requires bioactivation by cytochrome P450 (P450) enzymes to a pharmacologically active metabolite for antiplatelet action. The clinical limitations of clopidogrel are in large part due to its poor pharmacokinetics resulting from inefficient bioactivation by P450s. In this study, we determined the pharmacokinetics and pharmacodynamics of a novel conjugate of clopidogrel, referred to as ClopNPT, in animal models and we evaluated its potential to overcome the limitations of clopidogrel. Results from pharmacokinetic (PK) studies showed that ClopNPT released the active metabolite with a time to maximal plasma concentration of <5 minutes in C57BL/6 mice after either oral or intravenous administration, and plasma concentrations of the active metabolite reached Cmax values of 1242 and 1100 ng/ml after a 10-mg/kg oral dose and a 5-mg/kg intravenous dose, respectively. Furthermore, ClopNPT was highly effective in preventing arterial thrombosis in rabbits and mice after vascular injuries. Formation of occlusive thrombi was prevented by ClopNPT at the 1-mg/kg dose with no significant increase in tongue bleeding time, whereas clopidogrel was ineffective at the same dose. These results suggest that ClopNPT has favorable PK/pharmacodynamic properties that can potentially overcome the attenuated PK properties of clopidogrel and thus significantly improve the efficacy of antiplatelet therapy.
Assuntos
Artérias/efeitos dos fármacos , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Trombose/tratamento farmacológico , Ticlopidina/análogos & derivados , Animais , Artérias/fisiopatologia , Clopidogrel , Modelos Animais de Doenças , Fibrinolíticos/farmacocinética , Fibrinolíticos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Agregação Plaquetária/efeitos dos fármacos , Piridinas/química , Coelhos , Trombose/fisiopatologia , Ticlopidina/química , Ticlopidina/farmacocinética , Ticlopidina/farmacologia , Ticlopidina/uso terapêuticoRESUMO
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.
Assuntos
Androgênios/metabolismo , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Androstenodiona/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxilação , Cinética , Mutação , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Testosterona/metabolismoRESUMO
Hb is a protein with multiple functions, acting as an O2 transport protein, and having peroxidase and oxidase activities with xenobiotics that lead to substrate radicals. However, there is a lack of evidence for intermediates involved in these reactions of Hb with redox-active compounds, including those with xenobiotics such as drugs, chemical carcinogens, and sulfides. In particular, questions exist as to what intermediates participate in reactions of either metHb or oxyHb with sulfides. The studies presented here elaborate kinetics and intermediates involved in the reactions of Hb with oxidants (H2O2 and mCPBA), and they demonstrate the formation of high valent intermediates, providing insights into mechanistic issues of sulfur and drug oxidations. Overall, we propose generalized mechanisms that include peroxidatic reactions using H2O2 generated from the autooxidation of oxyHb, with involvement of substrate radicals in reactions of Hb with oxidizable drugs such as metyrapone or 2,4-dinitrophenylhydrazine and with sulfides. We identify ferryl intermediates (with a Soret band at 407 nm) in oxidative reactions with all of the above-mentioned reactions. These spectral properties are consistent with a protonated ferryl heme, such as Cpd II or Cpd ES-like species (Spolitak et al., JIB, 2006, 100, 2034-2044). Mechanism(s) of Hb oxidative reactions are discussed.
Assuntos
Hemoglobinas/química , Hemoglobinas/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Sulfetos/química , Sulfetos/metabolismo , Cinética , Oxirredução , OxigênioRESUMO
The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO. For all of the P450s tested, hydroxylation of d4IFO was improved and dechloroethylation was reduced as compared with nondeuterated IFO. Although the differences were not statistically significant, the trend favoring the 4'-hydroxylation pathway was noteworthy. CYP3A5 and CYP2C19 were the most efficient enzymes for catalyzing IFO hydroxylation. The importance of these enzymes in IFO metabolism has not been reported previously and warrants further investigation. The catalytic ability of the common polymorphisms of CYP2B6 and CYP2C9 for both reactions were tested with IFO and d4IFO. It was determined that the commonly expressed polymorphisms CYP2B6*4 and CYP2B6*6 had reduced catalytic ability for IFO compared with CYP2B6*1, whereas CYP2B6*7 and CYP2B6*9 had enhanced catalytic ability. As with the wild-type enzymes, d4IFO was more readily hydroxylated by the polymorphic variants than IFO, and d4IFO was not dechloroethylated by any of the polymorphic forms. We also assessed the use of specific inhibitors of P450 to favor hydroxylation in human liver microsomes. We were unable to separate the pathways with these experiments, suggesting that multiple P450s are responsible for catalyzing both metabolic pathways for IFO, which is not observed with the closely related drug cyclophosphamide.
Assuntos
Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacocinética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ifosfamida/metabolismo , Ifosfamida/farmacocinética , Animais , Catálise , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Deutério , Humanos , Hidroxilação , Técnicas In Vitro , Marcação por Isótopo , Microssomos Hepáticos/metabolismo , Polimorfismo Genético/genética , RatosRESUMO
Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro. Therefore, we characterized the MBI of CYP2B6 by CPS. CPS inactivated CYP2B6 in a time- and concentration-dependent manner with a kinact of 1.97 min(-1), a KI of 0.47 µM, and a partition ratio of 17.7. We further evaluated the ability of other organophosphate pesticides including chorpyrifos-methyl, diazinon, parathion-methyl, and azinophos-methyl to inactivate CYP2B6. These organophosphate pesticides were also potent MBIs of CYP2B6 characterized by similar kinact and KI values. The inactivation of CYP2B6 by CPS was accompanied by the loss of P450 detectable in the CO reduced spectrum and loss of detectable heme. High molecular weight aggregates were observed when inactivated CYP2B6 was run on SDS-PAGE gels indicating protein aggregation. Interestingly, we found that the rat homologue of CYP2B6, CYP2B1, was not inactivated by CPS despite forming CPO to a similar extent. On the basis of the locations of the Cys residues in the two proteins which could react with released sulfur during the metabolism of CPS, we investigated whether the C475 in CYP2B6, which is not conserved in CYP2B1, was the critical residue for inactivation by mutating it to a Ser. CYP2B6 C475S was inactivated to a similar extent as wild type CYP2B6 indicating that C475 is not likely the key difference between CYP2B1 and CYP2B6 with respect to inactivation. These results indicate that CPS and other organophosphate pesticides are potent MBIs of CYP2B6 which may have implications for the toxicity of these pesticides as well as the potential for pesticide-drug interactions.
Assuntos
Clorpirifos/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Inseticidas/metabolismo , Animais , Clorpirifos/química , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Eletroforese em Gel de Poliacrilamida , Heme/metabolismo , Humanos , Inseticidas/química , Cinética , Mutagênese Sítio-Dirigida , Ligação Proteica , Ratos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Citocromo P-450 CYP2E1/química , Peróxido de Hidrogênio/farmacologia , Antioxidantes/química , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2E1/genética , Inibidores do Citocromo P-450 CYP2E1 , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Heme/química , Mutação , NADP/químicaRESUMO
The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 µM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, ß1-ß2 loop, and ß4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Fenantrenos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Hidrocarboneto de Aril Hidroxilases/química , Domínio Catalítico , Cristalografia por Raios X , Família 2 do Citocromo P450 , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , CoelhosRESUMO
In this work, we investigated the formation, reactivity, and antiplatelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the 10 thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol, and 3-nitropyridine-2-thiol (NPT). The mass spectrometry (MS) and MS(2) spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M(-1)s(-1). The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pretreatment with 1 mM GSH, confirming that the AM is responsible for the antiplatelet activity of clopidogrel. Collectively, our results provide strong support for a cytochrome P450 (P450)-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate, followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored to overcome the interindividual variability in clopidogrel therapy.
Assuntos
Dissulfetos/química , Dissulfetos/farmacologia , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Ticlopidina/análogos & derivados , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Clopidogrel , Dissulfetos/metabolismo , Masculino , Inibidores da Agregação Plaquetária/metabolismo , Coelhos , Ticlopidina/química , Ticlopidina/metabolismo , Ticlopidina/farmacologiaRESUMO
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.
Assuntos
Anticarcinógenos/farmacologia , Anticarcinógenos/farmacocinética , Inibidores do Citocromo P-450 CYP2E1 , Isotiocianatos/farmacologia , Isotiocianatos/farmacocinética , Biotransformação , Humanos , Técnicas In Vitro , Isocianatos/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , OxirreduçãoRESUMO
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.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Catálise/efeitos dos fármacos , Inibidores do Citocromo P-450 CYP3A , Heme/metabolismo , Ritonavir/farmacologia , Apoproteínas/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/metabolismo , Glutationa/metabolismo , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismoRESUMO
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.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2E1/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Bases , Catálise , Família 2 do Citocromo P450 , Primers do DNA , Hidroxilação , Especificidade por SubstratoRESUMO
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.
Assuntos
Glucuronosiltransferase/metabolismo , Marcação por Isótopo , Espectrometria de Massas/métodos , Microssomos Hepáticos/enzimologia , Peptídeos/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Calibragem , Cromatografia Líquida , Estradiol/metabolismo , Glucuronídeos/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/química , Tripsina/metabolismoRESUMO
Arachidonoyl ethanolamide (anandamide) is an endogenous amide of arachidonic acid and an important signaling mediator of the endocannabinoid system. Given its numerous roles in maintaining normal physiological function and modulating pathophysiological responses throughout the body, the endocannabinoid system is an important pharmacological target amenable to manipulation directly by cannabinoid receptor ligands or indirectly by drugs that alter endocannabinoid synthesis and inactivation. The latter approach has the possible advantage of more selectivity, thus there is the potential for fewer untoward effects like those that are traditionally associated with cannabinoid receptor ligands. In that regard, inhibitors of the principal inactivating enzyme for anandamide, fatty acid amide hydrolase (FAAH), are currently in development for the treatment of pain and inflammation. However, several pathways involved in anandamide synthesis, metabolism, and inactivation all need to be taken into account when evaluating the effects of FAAH inhibitors and similar agents in preclinical models and assessing their clinical potential. Anandamide undergoes oxidation by several human cytochrome P450 (P450) enzymes, including CYP3A4, CYP4F2, CYP4X1, and the highly polymorphic CYP2D6, forming numerous structurally diverse lipids, which are likely to have important physiological roles, as evidenced by the demonstration that a P450-derived epoxide of anandamide is a potent agonist for the cannabinoid receptor 2. The focus of this review is to emphasize the need for a better understanding of the P450-mediated pathways of the metabolism of anandamide, because these are likely to be important in mediating endocannabinoid signaling as well as the pharmacological responses to endocannabinoid-targeting drugs.
Assuntos
Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Alcamidas Poli-Insaturadas/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Analgésicos não Narcóticos/metabolismo , Analgésicos não Narcóticos/farmacologia , Animais , Ácidos Araquidônicos/farmacologia , Moduladores de Receptores de Canabinoides/farmacologia , Canabinoides/metabolismo , Canabinoides/farmacologia , Cannabis/química , Desenho de Fármacos , Endocanabinoides , Humanos , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Ligantes , Fumar Maconha , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Oxirredução , Alcamidas Poli-Insaturadas/farmacologia , Receptores de Canabinoides/fisiologiaRESUMO
Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid metabolizing P450s and their corresponding products using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.
Assuntos
Sistema Enzimático do Citocromo P-450 , Endocanabinoides , Sistema Enzimático do Citocromo P-450/metabolismo , Endocanabinoides/metabolismo , Heme/metabolismo , Microssomos Hepáticos , Oxirredução , Proteínas Recombinantes/metabolismo , Xenobióticos/metabolismoRESUMO
We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839-847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) < l-Cys (53%) < ascorbic acid (61%) < GSH (100%). AM-thiol conjugates were observed in the presence of NAC, l-Cys, and GSH. In the case of GSH, the formation of both the AM and the glutathionyl conjugate was dependent on the GSH concentrations, with similar K(m) values of ~0.5 mM, which indicates that formation of the thiol conjugates constitutes an integral part of the bioactivation processes for clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ~10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo.
Assuntos
Microssomos Hepáticos/metabolismo , Compostos de Sulfidrila/metabolismo , Ticlopidina/análogos & derivados , Clopidogrel , Relação Dose-Resposta a Droga , Glutationa/farmacologia , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Oxirredução , Compostos de Sulfidrila/síntese química , Ticlopidina/síntese química , Ticlopidina/metabolismoRESUMO
Methadone is a µ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a K(I) = 10.0 µM and k(inact) = 0.027 min⻹. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone.