RESUMO
Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.
Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , ProteóliseRESUMO
The over two dozen CYP2B structures of human, rabbit, and woodrat enzymes solved in the last decade have significantly enhanced our understanding of the structure-function relationships of drug metabolizing enzymes. More recently, an important role has emerged for halogen-π interactions in the CYP2B6 active site in substrate selectivity, explaining in part the preference for halogenated ligands as substrates. The mechanism by which such ligands interact with CYP2B enzymes involves conserved phenylalanine side chains, in particular F108, F115, or F297, in the active site, which form π bonds with halogens. To illustrate such halogen-π interactions using drugs that are major substrates of CYP2B6, we present here a crystal structure of CYP2B6 in complex with an analog of the widely used anti-HIV drug efavirenz, which contains a methyl group in place of the carbonyl oxygen. The chlorine of the efavirenz analog forms a π bond with the aromatic ring of F108, whereas the putative metabolism site on the distal end of the molecule is oriented towards the heme iron. The crystal structure showcases how CYP2B6 accommodates this important drug analog of considerable size in the active site by movement of various side chains without substantially increasing the active site volume. Furthermore, the CYP2B6-efavirenz analog complex provides a useful platform to investigate computationally as well as biophysically the effect of genetic polymorphisms on binding of the widely studied efavirenz.
Assuntos
Benzoxazinas/química , Citocromo P-450 CYP2B6/química , Alcinos , Domínio Catalítico , Cristalografia por Raios X , Ciclopropanos , HumanosRESUMO
Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae), exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb-drug interaction (HDI) through cytochrome P450s (CYPs)-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs) and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki) values of 14.3, 16.8, 41.7, and 6.84 µM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone-drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.
Assuntos
Benzopiranos/química , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP3A/química , Dioxóis/química , Interações Ervas-Drogas , Saururaceae/química , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Fármacos Antiobesidade/química , Fármacos Antiobesidade/isolamento & purificação , Fármacos Antiobesidade/farmacologia , Benzopiranos/isolamento & purificação , Benzopiranos/farmacologia , Sítios de Ligação , Domínio Catalítico , Clorzoxazona/química , Clorzoxazona/farmacologia , Clopidogrel , Ciclobutanos/química , Ciclobutanos/farmacologia , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/isolamento & purificação , Inibidores das Enzimas do Citocromo P-450/farmacologia , Dioxóis/isolamento & purificação , Dioxóis/farmacologia , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Cinética , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Simulação de Acoplamento Molecular , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Ticlopidina/análogos & derivados , Ticlopidina/química , Ticlopidina/farmacologiaRESUMO
Chlorpyrifos (CPS) is a broad-spectrum organophosphate insecticide that is neurotoxic in humans. Chlorpyrifos oxon (CPO) is a toxic metabolite of CPS that is produced by CYP2B6. In this study, we examined the variability of CPS metabolism resulting from single-nucleotide polymorphisms in CYP2B6. Wild-type CYP2B6 (CYP2B6.1) and two variants each with a single amino acid substitution: CYP2B6.5 (R487C) and CYP2B6.8 (K139E) were co-expressed together with human NADPH-dependent cytochrome P450 reductase in Escherichia coli (E. coli). Both of the CYP2B6 variants were successfully expressed in E. coli. The conversion of CPS to CPO by the CYP2B6 variants was analyzed with high-performance liquid chromatography. Km and Vmax of the reaction by CYP2B6.1 were 18.50±2.94µM and 17.07±1.15mol/min/mol P450, respectively. The CYP2B6 variants produced CPO with the following kinetic parameters: Km for CYP2B6.5 and CYP2B6.8 were 20.44±6.43 and 44.69±9.97µM, respectively; and Vmax were 1.10±0.10 and 1.77±0.26mol/min/mol P450, respectively. These results indicate that the amino acid substitutions in the CYP2B6 variants suppressed the metabolic activation of CPS. CYP2B6 variants have altered capacity to bioactivate CPF and may affect individual susceptibility of CPF.
Assuntos
Clorpirifos/análogos & derivados , Citocromo P-450 CYP2B6/genética , Inseticidas/farmacocinética , Isoenzimas/genética , Polimorfismo de Nucleotídeo Único , Ativação Metabólica , Sequência de Aminoácidos , Substituição de Aminoácidos , Western Blotting , Clorpirifos/farmacocinética , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Homologia de Sequência de AminoácidosRESUMO
Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Citocromo P-450 CYP2B1/antagonistas & inibidores , Citocromo P-450 CYP2B6/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Éteres Difenil Halogenados/farmacologia , Alquilação/efeitos dos fármacos , Substituição de Aminoácidos , Compostos de Anilina , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Derivados de Benzeno/farmacologia , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Inibidores das Enzimas do Citocromo P-450/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Citocromos b5/metabolismo , Poluentes Ambientais/metabolismo , Éteres Difenil Halogenados/metabolismo , Humanos , Hidrocarbonetos Halogenados/metabolismo , Concentração Inibidora 50 , Estrutura Molecular , Mutagênese Sítio-Dirigida , NADPH Oxidases/metabolismo , Oxirredução , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Numerous cytochrome P450 (CYP) 2B6 substrates including drugs and environmental chemicals are halogenated. To assess the role of halogen-π bonds in substrate selectivity and orientation in the active site, structures of four CYP2B6 monoterpenoid complexes were solved by X-ray crystallography. Bornyl bromide exhibited dual orientations in the active site with the predominant orientation revealing a bromine-π bond with the Phe108 side chain. Bornane demonstrated two orientations with equal occupancy; in both, the C2 atom that bears the bromine in bornyl bromide was displaced by more than 2.5 Å compared with the latter complex. The bromine in myrtenyl bromide π-bonded with Phe297 in CYP2B6, whereas the two major orientations in the active site mutant I114V exhibited bromine-π interactions with two additional residues, Phe108 and Phe115. Analysis of existing structures suggests that halogen-π interactions may be unique to the CYP2B enzymes within CYP family 2 but are also important for CYP3A enzymes.
Assuntos
Domínio Catalítico , Citocromo P-450 CYP2B6/química , Halogênios/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Monoterpenos/química , Ligação Proteica , Especificidade por SubstratoRESUMO
Previously, we observed that the oxazinone ring is important for cytochrome P450 2B6 (CYP2B6) activity toward efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one), a CYP2B6 substrate used to treat HIV. To further understand the structural characteristics of efavirenz that render it a CYP2B6 substrate, we tested the importance of each heteroatom of the oxazinone ring. We assembled a panel of five analogues: 6-chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-2-methyl-4-(trifluoromethyl)-2H-3,1-benzoxazine (1), (4S)-6-chloro-4-[(1E)-2-cyclopropylethenyl]-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (2), (4S)-6-chloro-4-(2-cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (3), 6-chloro-4-(cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinolinone (4), and 6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-4H-benzo[d][1,3]dioxin-2-one (5). The metabolism of compounds 1-5 was investigated using human liver microsomes, individual P450s, and mass spectrometry or UV/Vis absorbance detection. Steady-state analysis of CYP2B6 metabolism of 1-5 showed KM values ranging from 0.3- to 3.9-fold different from that observed for efavirenz (KM : 3.6±1.7â µm). The lowest KM values, approximating 1â µm, were observed for the metabolism of 1, whereas the greatest KM value, 14±6.4â µm, was found for 4. Our work reveals that analogues with heteroatom changes in the oxazinone ring are still CYP2B6 substrates, although the changes in KM suggest altered substrate binding.
Assuntos
Benzoxazinas/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Alcinos , Benzoxazinas/análise , Benzoxazinas/química , Cromatografia Líquida de Alta Pressão , Ciclopropanos , Citocromo P-450 CYP2B6/química , Humanos , Cinética , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Cumarínicos/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Modelos Moleculares , Alquilação , Substituição de Aminoácidos , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Sítios de Ligação , Domínio Catalítico , Cumarínicos/química , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Família 2 do Citocromo P450 , Halogenação , Humanos , Cinética , Ligantes , Conformação Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. METHODS: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. RESULTS: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. CONCLUSIONS: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. GENERAL SIGNIFICANCE: The result provides a new basis for determining the structural elements that control functions in complex enzymes.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Cumarínicos/metabolismo , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Família 2 do Citocromo P450 , Cães , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esteroide Hidroxilases/química , Esteroide Hidroxilases/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: Tobacco smoking is a leading cause of preventable disease and death globally. Nicotine is the main addictive component in tobacco. Nicotine is eliminated from the body by biotransformation in the liver to inactive metabolites. This reaction is catalyzed by the cytochrome P450 2A6 (CYP2A6) enzyme. Administering chemical inhibitors of CYP2A6 has been shown to slow down the elimination of nicotine with consequent reduction in number of cigarettes smoked. We have systematically developed small molecule CYP2A6 inhibitors with good balance between potency and CYP selectivity. OBJECTIVE: During this process we have noticed that many potent CYP2A6 inhibitors also inhibit other human liver CYP forms, most notably CYP1A2 and CYP2B6. This study aimed at defining common and distinct features of ligand binding to CYP1A2, CYP2A6 and CYP2B6 active sites. METHODS: We used our previous chemical inhibitor databases to construct improved 3-dimensional quantitative structureactivity relationship (3D-QSAR) models for CYP1A2, CYP2A6 and CYP2B6. RESULTS: Combined 3D-QSAR and docking procedures yielded precise information about the common and distinct interactions of inhibitors and the enzyme active sites. Positioning of hydrogen bond donor/acceptor atoms and the shape and volume of the compound defined the potency and specificity of inhibition. A novel potent and selective CYP1A2 inhibitor was found. CONCLUSION: This in silico approach will provide a means for very rapid and high throughput prediction of cross-inhibition of these three CYP enzymes.
Assuntos
Inibidores do Citocromo P-450 CYP1A2/farmacologia , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2A6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2B6/metabolismo , Desenho de Fármacos , Domínio Catalítico , Desenho Assistido por Computador , Citocromo P-450 CYP1A2/química , Inibidores do Citocromo P-450 CYP1A2/química , Inibidores do Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2A6/química , Citocromo P-450 CYP2B6/química , Inibidores do Citocromo P-450 CYP2B6/química , Inibidores do Citocromo P-450 CYP2B6/metabolismo , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Relação Quantitativa Estrutura-AtividadeRESUMO
Furanocoumarin compound psoralen (PRN) is a major active ingredient found in herbaceous plants. PRN has been used for the treatment of various dermal diseases in China. We evaluated the inhibitory effect of PRN on cytochrome P450 2B6 (CYP2B6) and found that PRN induced a time-, concentration-, and NADPH-dependent inactivation of CYP2B6 with the values of KI and kinact being 110.2 µM and 0.200 min(-1), respectively. Ticlopidine, a CYP2B6 substrate, prevented the enzyme from the inactivation induced by PRN. Exogenous nucleophile glutathione (GSH) and catalase/superoxide dismutase showed limited protection of CYP2B6 from the inactivation. The estimated partition ratio of the inactivation was approximately 400. GSH trapping experiments indicates that an epoxide or/and γ-ketoenal intermediate was formed in microsomal incubations with PRN. In summary, PRN was characterized as a mechanism-based inactivator of CYP2B6.
Assuntos
Inibidores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2B6/metabolismo , Ficusina/farmacologia , Citocromo P-450 CYP2B6/química , Inibidores do Citocromo P-450 CYP2B6/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ficusina/química , Ticlopidina/metabolismo , Ticlopidina/farmacologiaRESUMO
The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.
Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Cumarínicos/química , Citocromo P-450 CYP2B6/química , Substituição de Aminoácidos , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Domínio Catalítico , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Família 2 do Citocromo P450 , Humanos , Mutação de Sentido Incorreto , OxirreduçãoRESUMO
The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.
Assuntos
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Aditivos Alimentares/metabolismo , Microssomos Hepáticos/enzimologia , Monoterpenos/metabolismo , Perfumes/metabolismo , Monoterpenos Acíclicos , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP2C19/genética , Inibidores das Enzimas do Citocromo P-450/farmacologia , Aditivos Alimentares/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidroxilação/efeitos dos fármacos , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Monoterpenos/química , Mariposas , Odorantes , Perfumes/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
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
Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5ß-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.
Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP3A/química , Éteres Difenil Halogenados/química , Ácido Litocólico/química , Animais , Catálise , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Éteres Difenil Halogenados/metabolismo , Humanos , Hidroxilação , Ácido Litocólico/metabolismo , Microssomos Hepáticos/enzimologia , Especificidade por SubstratoRESUMO
X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.
Assuntos
Citocromo P-450 CYP2A6/química , Citocromo P-450 CYP2B6/química , Monoterpenos/química , Compostos Orgânicos Voláteis/química , Monoterpenos Bicíclicos , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
The COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS) was used to predict the boiling points of several polybrominated diphenyl ethers (PBDEs) and methylated derivatives (MeO-BDEs) of monohydroxylated BDE (OH-BDE) metabolites. The linear correlation obtained by plotting theoretical boiling points calculated by COSMO-RS against experimentally determined retention times from gas chromatography-mass spectrometry facilitated the identification of PBDEs and OH-BDEs. This paper demonstrates the applicability of COSMO-RS in identifying unknown PBDE metabolites of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',6-pentabromodiphenyl ether (BDE-100). Metabolites of BDE-47 and BDE-100 were formed through individual incubations of each PBDE with recombinant cytochrome P450 2B6. Using calculated boiling points and characteristic mass spectral fragmentation patterns of the MeO-BDE positional isomers, the identities of the unknown monohydroxylated metabolites were proposed to be 2'-hydroxy-2,3',4,4'-tetrabromodiphenyl ether (2'-OH-BDE-66) from BDE-47, and 2'-hydroxy-2,3',4,4',6-pentabromodiphenyl ether (2'-OH-BDE-119) and 4-hydroxy-2,2',3,4',6-pentabromodiphenyl ether (4-OH-BDE-91) from BDE-100. The collective use of boiling points predicted with COSMO-RS, and characteristic mass spectral fragmentation patterns provided a valuable tool toward the identification of isobaric compounds.
Assuntos
Éteres Difenil Halogenados/análise , Éteres Difenil Halogenados/metabolismo , Espectrometria de Massas , Temperatura de Transição , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Teoria Quântica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solventes/química , Fatores de TempoRESUMO
The aim of this study was to further characterize the expression and function of human CYP2B6 in a recently generated CYP2A13/2B6/2F1-transgenic (TG) mouse model, in which CYP2B6 is expressed selectively in the liver. The inducibility of CYP2B6 by phenobarbital (PB) and dexamethasone (DEX), known inducers of CYP2B6 in human liver, was examined in the TG mice, as well as in TG/Cyp2abfgs-null (or "CYP2B6-humanized") mice. Hepatic expression of CYP2B6 mRNA and protein was greatly induced by PB or DEX treatment in both TG and TG/Cyp2abfgs-null mice. Function of the transgenic CYP2B6 was first studied using bupropion as a probe substrate. In PB-treated mice, the rates of hepatic microsomal hydroxybupropion formation (at 50 µM bupropion) were >4-fold higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice (for both male and female mice); the rate difference was accompanied by a 5-fold higher catalytic efficiency in the TG/Cyp2abfgs-null mice and was abolished by an antibody to CYP2B6. The ability of CYP2B6 to metabolize nicotine was then examined, both in vitro and in vivo. The rates of hepatic microsomal cotinine formation from nicotine were significantly higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice, pretreated with PB or DEX. Furthermore, systemic nicotine metabolism was faster in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice. Thus, the transgenic CYP2B6 was inducible and functional, and, in the absence of mouse CYP2A and CYP2B enzymes, it contributed to nicotine metabolism in vivo. The CYP2B6-humanized mouse will be valuable for studies on in vivo roles of hepatic CYP2B6 in xenobiotic metabolism and toxicity.
Assuntos
Indutores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2B6/metabolismo , Dexametasona/farmacologia , Fígado/efeitos dos fármacos , Nicotina/metabolismo , Fenobarbital/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Hipnóticos e Sedativos/farmacologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
In proteins with buried active sites, understanding how ligands migrate through the tunnels that connect the exterior of the protein to the active site can shed light on substrate specificity and enzyme function. A growing body of evidence highlights the importance of protein flexibility in the binding site on ligand binding; however, the influence of protein flexibility throughout the body of the protein during ligand entry and egress is much less characterized. We have developed a novel tunnel prediction and evaluation method named IterTunnel, which includes the influence of ligand-induced protein flexibility, guarantees ligand egress, and provides detailed free energy information as the ligand proceeds along the egress route. IterTunnel combines geometric tunnel prediction with steered molecular dynamics in an iterative process to identify tunnels that open as a result of ligand migration and calculates the potential of mean force of ligand egress through a given tunnel. Applying this new method to cytochrome P450 2B6, we demonstrate the influence of protein flexibility on the shape and accessibility of tunnels. More importantly, we demonstrate that the ligand itself, while traversing through a tunnel, can reshape tunnels due to its interaction with the protein. This process results in the exposure of new tunnels and the closure of preexisting tunnels as the ligand migrates from the active site.
Assuntos
Algoritmos , Citocromo P-450 CYP2B6/química , Imidazóis/química , Simulação de Dinâmica Molecular , Domínio Catalítico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , TermodinâmicaRESUMO
In this study, we investigated the influence of single nucleotide polymorphisms on the conformation of mutated cytochrome P450 (CYP) 2B6 proteins using molecular dynamics (MD) simulation. Some of these mutations influence drug metabolism activities, leading to individual variations in drug efficacy and pharmacokinetics. Using computational docking, we predicted the structure of the complex between the antimalarial agent artemether and CYP2B6 whose conformations were obtained by MD simulation. The simulation demonstrated that the entire structure of the protein changes even when a single residue is mutated. Moreover, the structural flexibility is affected by the mutations and it may influence the enzyme activity. The results suggest that some of the inactive mutants cannot recognize artemether due to structural changes caused by the mutation.