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1.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480231

RESUMO

Human cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6',7'-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Furocumarinas/química , Furocumarinas/metabolismo , Mibefradil/química , Mibefradil/metabolismo , Triazóis/química , Triazóis/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Especificidade por Substrato
2.
Chem Pharm Bull (Tokyo) ; 67(11): 1183-1190, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31423003

RESUMO

For rational drug design, it is essential to predict the binding mode of protein-ligand complexes. Although various machine learning-based models have been reported that use convolutional neural networks (deep learning) to predict binding modes from three-dimensional structures, there are few detailed reports on how best to construct and use datasets. Here, we examined how different datasets affected the prediction of the binding mode of CYP3A4 by a three-dimensional neural network when the number of crystal structures for the target protein was limited. We used four different training datasets: one large, general dataset containing various protein complexes and three smaller, more specific datasets containing complexes with CYP3A4-like pockets, complexes with CYP3A4-binding ligands, and complexes with CYP protein family members. We then trained models with different combinations of datasets with or without subsequent fine-tuning and evaluated the binding mode prediction performance of each model. The best receiver operating characteristic (ROC) area under the curve (AUC) model with respect to area under the receiver operating characteristic curve was obtained by training with a combination of the general protein and CYP family datasets. However, the ROC AUC-recall balanced model was obtained by training with this combination of datasets followed by fine-tuning with the CYP3A4-binding ligands dataset. Our results suggest that datasets that balance protein functionality and data size are important for optimizing binding mode prediction performance. In addition, datasets with large median binding pocket sizes may be important for the binding mode prediction specifically of CYP3A4.


Assuntos
Citocromo P-450 CYP3A/química , Aprendizado Profundo , Sítios de Ligação , Citocromo P-450 CYP3A/metabolismo , Bases de Dados Factuais , Humanos , Ligantes
3.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295928

RESUMO

The metabolism of vandetanib, a tyrosine kinase inhibitor used for treatment of symptomatic/progressive medullary thyroid cancer, was studied using human hepatic microsomes, recombinant cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). The role of CYPs and FMOs in the microsomal metabolism of vandetanib to N-desmethylvandetanib and vandetanib-N-oxide was investigated by examining the effects of CYP/FMO inhibitors and by correlating CYP-/FMO-catalytic activities in each microsomal sample with the amounts of N-desmethylvandetanib/vandetanib-N-oxide formed by these samples. CYP3A4/FMO-activities significantly correlated with the formation of N-desmethylvandetanib/ vandetanib-N-oxide. Based on these studies, most of the vandetanib metabolism was attributed to N-desmethylvandetanib/vandetanib-N-oxide to CYP3A4/FMO3. Recombinant CYP3A4 was most efficient to form N-desmethylvandetanib, while FMO1/FMO3 generated N-oxide. Cytochrome b5 stimulated the CYP3A4-catalyzed formation of N-desmethylvandetanib, which is of great importance because CYP3A4 is not only most efficient in generating N-desmethylvandetanib, but also most significant due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib. Indeed, the CYP3A4-mediated reaction exhibits kinetics of positive cooperativity and this corresponded to the in silico model, where two vandetanib molecules were found in CYP3A4-active center.


Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Enzimas/metabolismo , Oxirredução , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Animais , Antineoplásicos/química , Citocromo P-450 CYP3A/química , Relação Dose-Resposta a Droga , Enzimas/química , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Piperidinas/química , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Coelhos , Ratos , Proteínas Recombinantes
4.
Aquat Toxicol ; 214: 105239, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31280135

RESUMO

CYP3A enzymes play a crucial role in metabolic clearance of a variety of xenobiotics. However, their genetic information and function remain unclear in molluscs. In the present study, two novel CYP3A genes i.e. McCYP3A-1 and McCYP3A-2 were identified and characterized from the thick shell mussel Mytilus coruscus, and their tissue distribution as well as the response to cadmium (Cd) and benzo[a]pyrene (B[α]P) exposure were addressed using real time quantitative RT-PCR (qRT-PCR) and erythromycin N-demethylase (ERND) assay. McCYP3A-1 and McCYP3A-2 possess typically domains of CYP family such as helix-C, helix-I, helix-K, PERF and the heme binding domain as well as the characteristic domains of CYP3s including six SRS motifs. McCYP3A-1 and McCYP3A-2 transcripts were constitutively expressed in all examined tissues with high expression level in digestive glands, hepatopancreas and gonads. Upon B[α]P exposure, McCYP3A-1 and McCYP3A-2 mRNA expression in digestive glands showed a pattern of up-regulation followed by down-regulation, while under Cd exposure, showed a time-dependent induction profile. In addition, ERND activity, generally used as an indicator of CYP3, increased in a time-dependent manner after exposure to Cd and B[α]P. These results collectively indicated that McCYP3A-1 and McCYP3A-2 are CYP3A family member and may play a potential role in metabolic clearance of xenobiotics. Meanwhile, the current results may provide some baseline data to support McCYP3A-1 and McCYP3A-2 as candidate biomarkers for monitoring of PAHs and heavy metal pollution.


Assuntos
Organismos Aquáticos/enzimologia , Benzo(a)pireno/toxicidade , Cádmio/toxicidade , Citocromo P-450 CYP3A/metabolismo , Mytilus/enzimologia , Sequência de Aminoácidos , Animais , Organismos Aquáticos/efeitos dos fármacos , Sequência de Bases , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mytilus/efeitos dos fármacos , Filogenia , Distribuição Tecidual/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade
5.
Biosens Bioelectron ; 135: 160-165, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31009883

RESUMO

In the present work, a double photoelectrode system has been constructed for photoelectrochemically driven enzymatic bioconversion and determination of nifedipine. In which, the TiO2 nanotube arrays in-situ assembled with g-C3N4 (TNA/g-C3N4) was used as a photoanode, and a cytochrome P450 3A4 (CYP3A4) enzyme was immobilized in the porous ITO/CuO films to fabricate an ITO/CuO/CYP3A4 photocathode. The constructed double photoelectrode system had a significant photocurrent response compared to the single ITO/CuO/CYP3A4 or TNA/g-C3N4 under visible light irradiation. Under optimal conditions, the photocurrent of the double photoelectrode system had a high catalytic activity toward substrate nifedipine with kcat of 5.62 s-1 and catalytic efficiency with kcat/kmapp of 0.94 µM-1 s-1, and the bioconversion yield of nifedipine reached 22.1%. Furthermore, the constructed double photoelectrode system could be used to determine the nifedipine concentration with a high sensitivity of 2.46 µA µM-1 and a low detection limit of 0.015 µM. Therefore, the proposed double photoelectrode system can be used well for study enzyme biocatalysis for target bioconversion, and also has a potential application for toxicity analysis.


Assuntos
Nifedipino/análise , Vasodilatadores/análise , Biocatálise , Técnicas Biossensoriais/instrumentação , Citocromo P-450 CYP3A/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Luz , Nanotubos/química , Nanotubos/ultraestrutura , Titânio/química
6.
PLoS One ; 14(3): e0214338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908543

RESUMO

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.


Assuntos
Clonagem Molecular/métodos , Citocromo P-450 CYP3A/genética , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Sítios de Ligação , Bovinos , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Transfecção
7.
J Biol Chem ; 294(20): 8015-8022, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30926609

RESUMO

Cytochrome P450 (CYP) 3A4 is a major contributor to hepatic drug and xenobiotic metabolism in human adults. The related enzyme CYP3A5 is also expressed in adult liver and has broader age and tissue distributions. However, CYP3A5 expression is low in most Caucasians because of the prevalence of an allele that leads to an incorrectly spliced mRNA and premature termination of translation. When expressed, CYP3A5 expands metabolic capabilities and can augment CYP3A4-mediated drug metabolism, thereby reducing drug efficacy and potentially requiring dose adjustments. The extensive role of CYP3A4 in drug metabolism reflects in part the plasticity of the substrate-free enzyme to enlarge its active site and accommodate very large substrates. We have previously shown that the structure of the CYP3A5-ritonavir complex differs substantially from that of the CYP3A4-ritonavir complex. To better understand whether these differences are conserved in other CYP3A5 structures and how they relate to differential plasticity, we determined the X-ray crystallographic structure of the CYP3A5 substrate-free complex to 2.20 Å resolution. We observed that this structure exhibits a much larger active site than substrate-free CYP3A4 and displays an open substrate access channel. This reflected in part a lower trajectory of the helix F-F' connector in CYP3A4 and more extensive π-CH interactions between phenylalanine residues forming the roof of the active-site cavity than in CYP3A5. Comparison with the CYP3A5-ritonavir complex confirmed conserved CYP3A5 structural features and indicated differences in plasticity between CYP3A4 and CYP3A5 that favor alternative ritonavir conformations.


Assuntos
Citocromo P-450 CYP3A/química , Ritonavir/química , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP3A/metabolismo , Humanos
8.
Int J Mol Sci ; 20(4)2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30823507

RESUMO

We computed the network of channels of the 3A4 isoform of the cytochrome P450 (CYP) on the basis of 16 crystal structures extracted from the Protein Data Bank (PDB). The calculations were performed with version 2 of the CCCPP software that we developed for this research project. We identified the minimal cost paths (MCPs) output by CCCPP as probable ways to access to the buried active site. The algorithm of calculation of the MCPs is presented in this paper, with its original method of visualization of the channels. We found that these MCPs constitute four major channels in CYP3A4. Among the many channels proposed by Cojocaru et al. in 2007, we found that only four of them open in 3A4. We provide a refined description of these channels together with associated quantitative data.


Assuntos
Citocromo P-450 CYP3A/química , Algoritmos , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Humanos , Domínios Proteicos , Estrutura Quaternária de Proteína , Software
9.
J Mol Model ; 25(3): 65, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30762124

RESUMO

Soft spot analysis helps evaluate the site of the metabolic lability that impacts the bio-availability of the drug. However, given its laborious and time consuming experimentation, we propose a reliable and cheap in silico strategy. In this context, we hypothesized a mechanistic rationale for metabolism of erlotinib by the CYP3A4 enzyme. The comparison of the 3D conformations of the target CYP class of enzymes using MD simulations with GROMACS helped evaluate its impact on the metabolism. The molecular docking studies using Autodock-Vina ascertained the explicit role of the Fe ion present in the Heme moiety in this process. This mechanism was confirmed with respect to 13 other popular approved FDA kinase inhibitors using ab initio DFT calculations using Gaussian 09 (G09), molecular docking studies with Autodock-Vina, and MD simulations with GROMACS. We then developed a quantitative (Q-Met) metabolic profile of these soft spots in the molecules and demonstrated the lack of a linear relationship between the extent of metabolism and drug efficacy. We thus propose an economic in silico strategy for the early prediction of the lability in kinase inhibitors to help model their bio-availability and activity simultaneously, prior to clinical testing.


Assuntos
Citocromo P-450 CYP3A/química , Cloridrato de Erlotinib/farmacocinética , Inibidores de Proteínas Quinases/química , Sítios de Ligação , Disponibilidade Biológica , Simulação por Computador , Citocromo P-450 CYP3A/metabolismo , Desenho de Drogas , Cloridrato de Erlotinib/química , Cloridrato de Erlotinib/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Termodinâmica
10.
Drug Metab Pharmacokinet ; 34(2): 113-125, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30639283

RESUMO

A hexagonal-grid based template system has been developed to a predicting tool of CYP3A4-mediated reactions through the reconstitution of the active site with the assembly of the ligands. Simultaneous interactions of flattened-shape ligands at two sites of CYP3A4, oxidizing- and triggering-sites, are essential ideas, which were supported in the simulation results of various ligands on the template. The interactions were accomplished with either uni-molecule bindings or bi-molecule bindings with ligands termed pro-metabolized and trigger molecules. The template shape was determined mainly through reciprocal comparisons of simulation results with available experiment data mainly on recombinant CYP3A4-mediated reactions of polyaromatic hydrocarbon (PAH) ligands. Through the applications of various PAH and non-PAH ligands on the template, couple region-specific interactions including mechanisms to facilitate ligand movement from the initial site to a place near heme-oxygen and to trigger of catalyses are envisioned. These are very effective tools to verify candidates of CYP3A4 ligands as the good or poor substrates. The bi-molecule binding idea also explains so called "cooperative bindings with activator/effector" as interactions with non-identical trigger molecules on this CYP3A4-template system, and also offers possible mechanisms of the non-linear kinetic behavior.


Assuntos
Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Inibidores das Enzimas do Citocromo P-450/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/metabolismo , Humanos , Cinética , Ligantes , Estrutura Molecular , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Proteínas Recombinantes/metabolismo
11.
Biochemistry ; 58(7): 930-939, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30676743

RESUMO

Binding of small inhibitory compounds to human cytochrome P450 3A4 (CYP3A4) could interfere with drug metabolism and lead to drug-drug interactions, the underlying mechanism of which is not fully understood due to insufficient structural information. This study investigated the interaction of recombinant CYP3A4 with a nonspecific inhibitor metyrapone, antifungal drug fluconazole, and protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Metyrapone and fluconazole are classic type II ligands that inhibit CYP3A4 with medium strength by ligating to the heme iron, whereas PMSF, lacking the heme-ligating moiety, acts as a weak type I ligand and inhibitor of CYP3A4. High-resolution crystal structures revealed that the orientation of metyrapone is similar but not identical to that in the previously reported 1W0G model, whereas the flexible fluconazole adapts a conformer markedly different from that observed in the target CYP51 enzymes, which could explain its high potential for cross-reactivity. Besides hydrophobic and aromatic interactions with the heme and active site residues, both drugs establish water-mediated contacts that stabilize the inhibitory complexes. PMSF also binds near the catalytic center, with the phenyl group parallel to the heme. However, it does not displace the water ligand and is held in place via strong H-bonds formed by the sulfofluoride moiety with Ser119 and Arg212. Collectively, our data suggest that PMSF might have multiple binding sites and likely occupies the high-affinity site in the crystal structure. Moreover, its hydrolysis product, phenylmethanesulfonic acid, can also access and be retained in the CYP3A4 active site. Therefore, to avoid experimental artifacts, PMSF should be excluded from purification and assay solutions.


Assuntos
Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP3A/farmacologia , Fluconazol/química , Fluconazol/metabolismo , Fluconazol/farmacologia , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Inativação Metabólica , Metirapona/química , Metirapona/metabolismo , Metirapona/farmacologia , Fluoreto de Fenilmetilsulfonil/química , Fluoreto de Fenilmetilsulfonil/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Serina/química , Serina/metabolismo
12.
Talanta ; 196: 231-236, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683357

RESUMO

We used rapid one-step derivatization of 6ß-hydroxylated hydrocortisone by sulfuric acid for fluorimetric determination of CYP3A4-dependent hydroxylase reaction in the electrochemical system. We have shown that CYP3A4 substrate - hydrocortisone - and its 6ß-hydroxylated product have different emission wavelengths at an excitation λex = 365 nm after treatment with sulfuric acid:ethanol (3:1) mixture (λem = 525 ±â€¯2 nm and λem = 427 ±â€¯2 nm, respectively). The detection limit for 6ß-hydroxycortisol was estimated to be 0.32 µM (corresponding to 0.095 nmol in 300 µL sample) (S/N = 3). Using the fluorimetric method of 6ß-hydroxycortisol detection following the electrolysis of hydrocortisone with CYP3A4 immobilized on a screen-printed graphite electrode modified by didodecyldimethylammonium bromide we have calculated the steady-state kinetic parameters of CYP3A4 for hydrocortisone: the maximal rate of the reaction (Vmax) as 89 ±â€¯5 pmol of product per min per pmol of electroactive enzyme and the Michaelis constant (KM) as 10 ±â€¯2 µM. In our system, ketoconazole inhibited hydroxylase activity of CYP3A4 towards hydrocortisone with the IC50 value of 70 ±â€¯5 nM. The approach proposed for determination of the CYP3A4 electrocatalytic activity can be used for throughput screening of different modulators of this cytochrome P450 isozyme during drug development.


Assuntos
Citocromo P-450 CYP3A/química , Enzimas Imobilizadas/química , Hidrocortisona/análogos & derivados , Hidrocortisona/química , Ácidos Sulfúricos/química , Catálise , Eletrólise , Fluorometria
13.
Food Chem Toxicol ; 123: 225-232, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30385190

RESUMO

The chiral pesticide fipronil is employed as a racemic mixture to control pests. Although there are no enantioselective differences in the fipronil enantiomer activities toward target organisms, fipronil enantiomers may exhibit enantioselective differences in their bioaccumulation, toxicity, and metabolism toward non-target organisms, including humans. The present work aims to provide significant reliable enantioselective information concerning fipronil risk assessment in humans. For that, the in vitro metabolism of rac-fipronil, S-fipronil, and R-fipronil by human liver microsomes was evaluated, the in vivo enantioselective toxicokinetic parameters were predicted and the main CYP450 isoforms involved in the enantioselective metabolism were determined. The obtained results demonstrated that fipronil may undergo a clearance by the liver and it is exclusively metabolized by the CYP3A4 isoform. Although no significative stereoselective differences were observed, the results provide reliable information on fipronil risk assessment for humans.


Assuntos
Praguicidas/química , Praguicidas/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Praguicidas/toxicidade , Pirazóis/toxicidade , Medição de Risco , Estereoisomerismo , Toxicocinética
14.
PLoS One ; 13(11): e0206279, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30427871

RESUMO

Two chemotypes were examined in vitro with CYPs 3A4 and 2C19 by molecular docking, metabolic profiles, and intrinsic clearance deuterium isotope effects with specifically deuterated form to assess the potential for enhancement of pharmacokinetic parameters. The results show the complexity of deuteration as an approach for pharmacokinetic enhancement when CYP enzymes are involved in metabolic clearance. With CYP3A4 the rate limiting step was chemotype-dependent. With one chemotype no intrinsic clearance deuterium isotope effect was observed with any deuterated form, whereas with the other chemotype the rate limiting step was isotopically sensitive, and the magnitude of the intrinsic clearance isotope effect was dependent on the position(s) and extent of deuteration. Molecular docking and metabolic profiles aided in identifying sites for deuteration and predicted the possibility for metabolic switching. However, the potential for an isotope effect on the intrinsic clearance cannot be predicted and must be established by examining select deuterated versions of the chemotypes. The results show how in a deuteration strategy molecular docking, in-vitro metabolic profiles, and intrinsic clearance assessments with select deuterated versions of new chemical entities can be applied to determine the potential for pharmacokinetic enhancement in a discovery setting. They also help explain the substantial failures reported in the literature of deuterated versions of drugs to elicit a systemic enhancement on pharmacokinetic parameters.


Assuntos
Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP3A/química , Deutério/química , Farmacocinética , Citocromo P-450 CYP2C19/efeitos da radiação , Citocromo P-450 CYP3A/efeitos da radiação , Deutério/farmacologia , Heme/química , Heme/efeitos da radiação , Humanos , Inativação Metabólica , Cinética , Microssomos/efeitos da radiação , Simulação de Acoplamento Molecular , Oxirredução/efeitos da radiação , Especificidade por Substrato
15.
ACS Appl Mater Interfaces ; 10(49): 41956-41961, 2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30422622

RESUMO

Recently, the early screening of the genotoxicity of new chemicals and drugs calls for the envelope of micro-/nanoreactors for metabolic study. Herein, a novel light-driven enzymatic bionanoreactor is designed with the gold nanoparticle (NP)-modified carbon nanocage (Au@CNC) as a nanoreactor and meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a photosensitizer for cytochrome P450-mediated drug metabolism. By confining the cytochrome P450 3A4 (CYP3A4) enzyme and TCPP inside the pores of Au@CNC, a high metabolic activity is achieved by using 7-ethoxytrifluoromethyl coumarin as the substrate because of the three-dimensional hierarchical porous structure, large surface area, and fast electron transfer capacity of Au@CNC. It is noted that owing to the presence of AuNPs inside CNC, the surface hydrophilicity of CNC is much improved, which further promotes the catalytic activity of the CYP3A4 enzyme. To our knowledge, this is the first attempt to apply CNC as a bionanoreactor for NADPH-free and light-driven in vitro drug metabolism. In addition, the presented bionanoreactor exhibits a variety of advantages in terms of fast response, short assay time (10 min), high sensitivity, and good selectivity, which are expected to expedite drug screening and render potential advances in drug discovery and development.


Assuntos
Carbono/química , Citocromo P-450 CYP3A/química , Enzimas Imobilizadas/química , Ouro/química , Nanopartículas Metálicas/química , Transporte de Elétrons , Humanos
16.
Molecules ; 23(10)2018 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-30301254

RESUMO

Flavonoids are natural compounds that have been extensively studied due to their positive effects on human health. There are over 4000 flavonoids found in higher plants and their beneficial effects have been shown in vitro as well as in vivo. However, data on their pharmacokinetics and influence on metabolic enzymes is scarce. The aim of this study was to focus on possible interactions between the 30 most commonly encountered flavonoid aglycones on the metabolic activity of CYP3A4 enzyme. 6ß-hydroxylation of testosterone was used as marker reaction of CYP3A4 activity. Generated product was determined by HPLC coupled with diode array detector. Metabolism and time dependence, as well as direct inhibition, were tested to determine if inhibition was reversible and/or irreversible. Out of the 30 flavonoids tested, 7 significantly inhibited CYP3A4, most prominent being acacetin that inhibited 95% of enzyme activity at 1 µM concentration. Apigenin showed reversible inhibition, acacetin, and chrysin showed combined irreversible and reversible inhibition while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results alert on possible flavonoid⁻drug interactions on the level of CYP3A4.


Assuntos
Inibidores do Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/efeitos dos fármacos , Flavonas/química , Flavonoides/química , Apigenina/química , Apigenina/farmacologia , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/farmacologia , Interações de Medicamentos , Flavanonas/química , Flavanonas/farmacologia , Flavonas/farmacologia , Flavonoides/farmacologia , Humanos , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacologia , Testosterona/química
17.
J Biol Chem ; 293(43): 16623-16634, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30217815

RESUMO

Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Metaloporfirinas/metabolismo , Metais/metabolismo , Protoporfirinas/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Citocromo P-450 CYP3A/química , Humanos , Metaloporfirinas/química , Dobramento de Proteína , Protoporfirinas/química , Esteroide 17-alfa-Hidroxilase/química , Esteroide 21-Hidroxilase/química
18.
Chem Res Toxicol ; 31(10): 1052-1060, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30203651

RESUMO

1,3,8-Trihydroxy-6-methylanthraquinone (emodin), a widely existing natural product in herbal medicines, has been reported to be hepatotoxic, but the exact underlying mechanism is still not fully understood. The objective of the present study was to evaluate the role of CYP3A and glutathione (GSH) in emodin-induced liver injury. Primary human hepatocytes were exposed to emodin with and without addition of CYP3A inducer/inhibitor and GSH synthesis inhibitor. It was found that emodin-mediated cytotoxicity increased when CYP3A was activated and GSH was depleted. Hepatotoxicity induced by emodin in rats by activation/inhibition of CYP3A and depletion of GSH was further investigated. Administration of emodin in combination with l-buthionine sulfoximine (BSO) or dexamethasone (DEX) resulted in aggravated liver injury, whereas pretreatment with ketoconazole (KTZ) suppressed the side effects caused by emodin. In addition, plasma exposure of emodin and its glucuronide metabolite were measured by ultraperformance liquid chromatography triple quadrupole mass spectrometry. Emodin and its glucuronide were lower in BSO-, DEX-, and KTZ- co-treated rats compared with those administered with emodin alone. In conclusion, these mentioned results suggested that CYP3A induction and GSH depletion might be involved in hepatotoxicity induced by emodin. This study may help to understand the risk factors and the mechanism of hepatotoxicity of emodin in humans.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Emodina/toxicidade , Glutationa/metabolismo , Animais , Butionina Sulfoximina/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/toxicidade , Dexametasona/toxicidade , Emodina/análise , Emodina/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley
19.
Chem Biol Interact ; 293: 115-123, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30086269

RESUMO

Metabolism of most endogenous and exogenous compounds is usually produced by the oxidation of cytochrome P450. Due to drug-drug interactions caused by the inhibition or induction of cytochrome P450 enzymes, changes in drug metabolism are the major causes of drug toxicity, CYP3A4 is one of the key isozymes, and involved in the metabolism of over 60% of clinical drugs. Human ether-a-go-go related genes (hERG) potassium channel is the most important target of many drugs and plays an important role in cardiac repolarization. Blockade of this channel may lead to long QT syndrome (LQTS), leading to sudden cardiac death. Therefore, it is necessary to evaluate the inhibitory properties of drugs on cytochrome P450 enzymes and hERG channel. We primarily evaluate the safety of berberine in combination with statins. Based on these findings, berberine in combination with statins has a greater inhibitory effect on CYP3A4 activity and CYP3A4 protein and mRNA expression than berberine alone. Simvastatin and atorvastatin reduce hERG current by accelerating channel inactivation. At the same time, the inhibitory effect of berberine and statin combination increased on hERG current by reducing the time constant of inactivation than the single drug alone. These results indicate that berberine in combination with statins can increase cardiotoxicity by inhibiting CYP3A4 and hERG channel.


Assuntos
Berberina/farmacologia , Citocromo P-450 CYP3A/metabolismo , Canal de Potássio ERG1/metabolismo , Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Atorvastatina/farmacologia , Atorvastatina/uso terapêutico , Berberina/uso terapêutico , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/genética , Células HEK293 , Células Hep G2 , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Cinética , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/patologia , Microssomos Hepáticos/metabolismo , Ratos , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Testosterona/metabolismo
20.
J Inorg Biochem ; 188: 9-17, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30098472

RESUMO

The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megaterium BM3 (BMR). Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. Vmax values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR Vmax values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem.


Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/química , Citocromo P-450 CYP3A/química , Sistema Enzimático do Citocromo P-450/química , Técnicas Eletroquímicas , NADPH-Ferri-Hemoproteína Redutase/química , Proteínas Recombinantes de Fusão/química , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Catálise , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética
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