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1.
Protein Sci ; 33(10): e5165, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39291728

RESUMO

Cytochrome P450 2B4 (CYP 2B4) is one of the best-characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4's globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzyme's interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Família 2 do Citocromo P450 , Simulação de Dinâmica Molecular , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Conformação Proteica , Coelhos
2.
J Inorg Biochem ; 259: 112667, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39032346

RESUMO

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Família 2 do Citocromo P450 , Mononucleotídeo de Flavina , NADPH-Ferri-Hemoproteína Redutase , NADPH-Ferri-Hemoproteína Redutase/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Mononucleotídeo de Flavina/metabolismo , Mononucleotídeo de Flavina/química , Família 2 do Citocromo P450/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/química , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/genética , Humanos , Mutagênese Sítio-Dirigida , Domínios Proteicos , Cinética , Animais
3.
Biophys Chem ; 301: 107092, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37586236

RESUMO

The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromos b5 , Citocromos b5/química , Citocromos b5/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Oxirredução
4.
J Biomol Struct Dyn ; 41(9): 4081-4092, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-35427216

RESUMO

CYP2A6 is a very important enzyme that plays a crucial role in nicotine compounds and is responsible for the metabolism of more than 3% drugs of total metabolized drugs by the CYP family and reported as one of very important pharmacogenes. CYP2A6 is highly polymorphic in nature and reported with more than 40 variants, most of these variants are SNPs originated and population specific. It has been well observed and reported that the presence of these population-specific non-synonymous SNPs in CYP2A6 alters the rate of drug metabolism and as a functional consequence, drugs produce an abnormal response. Though genomics and pharmacogenomics studies are there, very less is known about the structural effects of these SNPs on molecular-interaction and folding of CYP2A6. To fill the knowledge gap, SNPs based four variants, i.e., CYP2A6*2, CYP2A6*18, CYP2A6*21, and CYP2A6*35, which are frequently reported in the South Asian population, were considered for the study. Coumarin (DB04665), a well reported drug, is considered as a model substance, and the effect of all four variants on 'CYP2A6*-coumarin' complex was studied. MD simulation-based analysis (at 200 ns) was performed and comparative analysis with respect to wild type 'CYP2A6-coumarin' complex was done. Though observation didn't find any global effect on complete complex but found some crucial minor-local alteration in interaction and folding process. It is assumed that the change due to SNPs in the single amino acid did not bring global change in physiochemical properties of CYP2A6* but caused local-trivial changes which are very crucial for its metabolic activity.Communicated by Ramaswamy H. Sarma.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP2A6 , Oxigenases de Função Mista , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cumarínicos , Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Nicotina/metabolismo
5.
J Biol Chem ; 296: 100645, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839156

RESUMO

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Polímeros/metabolismo , Propilaminas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Catálise , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Coelhos
6.
Appl Biochem Biotechnol ; 193(1): 218-237, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32869209

RESUMO

We report the synthesis of seven new proluciferins for convenient activity determination of enzymes belonging to the cytochrome P450 (CYP) 4 family. Biotransformation of these probe substrates was monitored using each of the twelve human CYP4 family members, and eight were found to act at least on one of them. For all substrates, activity of CYP4Z1 was always highest, while that of CYP4F8 was always second highest. Site of metabolism (SOM) predictions involving SMARTCyp and docking experiments helped to rationalize the observed activity trends linked to substrate accessibility and reactivity. We further report the first homology model of CYP4F8 including suggested substrate recognition residues in a catalytically competent conformation accessed by replica exchange solute tempering (REST) simulations.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Família 4 do Citocromo P450/química , Tiazóis/química , Catálise , Humanos , Especificidade por Substrato
7.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375250

RESUMO

Prenatal smoke exposure (PreSE) is a risk factor for nicotine dependence, which is further enhanced by postnatal smoke exposure (PostSE). One susceptibility gene to nicotine dependence is Cytochrome P450 (CYP) 2A6, an enzyme responsible for the conversion of nicotine to cotinine in the liver. Higher CYP2A6 activity is associated with nicotine dependence and could be regulated through DNA methylation. In this study we investigated whether PostSE further impaired PreSE-induced effects on nicotine metabolism, along with Cyp2a5, orthologue of CYP2A6, mRNA expression and DNA methylation. Using a mouse model where prenatally smoke-exposed adult offspring were exposed to cigarette smoke for 3 months, enzyme activity, mRNA levels, and promoter methylation of hepatic Cyp2a5 were evaluated. We found that in male offspring, PostSE increased PreSE-induced cotinine levels and Cyp2a5 mRNA expression. In addition, both PostSE and PreSE changed Cyp2a5 DNA methylation in male groups. PreSE however decreased cotinine levels whereas it had no effect on Cyp2a5 mRNA expression or methylation. These adverse outcomes of PreSE and PostSE were most prominent in males. When considered in the context of the human health aspects, the combined effect of prenatal and adolescent smoke exposure could lead to an accelerated risk for nicotine dependence later in life.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Metabólica , Nicotina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Fumaça/efeitos adversos , Animais , Animais Recém-Nascidos , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Regiões Promotoras Genéticas
8.
J Chem Inf Model ; 60(2): 766-776, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-31622091

RESUMO

Tobacco smoke contains various cancer-causing toxic substances, including nicotine and nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). The cytochrome 2A13 is involved in nicotine metabolism and in the activation of the pro-carcinogenic agents NNK and NNN, by means of α-hydroxylation reactions. Despite the significance of cytochrome 2A13 in the biotransformation of these molecules, its conformational mechanism and the molecular basis involved in the process are not fully understood. In this study, we used molecular dynamics and principal component analysis simulations for an in-depth analysis of the essential protein motions involved in the interaction of cytochrome 2A13 with its substrates. We also evaluated the interaction of these substrates with the amino acid residues in the binding pocket of cytochrome 2A13. Furthermore, we quantified the nature of these chemical interactions from free energy calculations using the Molecular Mechanics/Generalized Born Surface Area method. The ligands remained favorably oriented toward compound I (cytochrome P450 O═FeIV state), to undergo α-hydroxylation. The hydrogen bond with asparagine 297 was essential to maintaining the substrates in a favorable catalytic orientation. The plot of first principal motion vs second principal motion revealed that the enzyme's interaction with nicotine and NNK involved different conformational subgroups, whereas the conformational subgroups in the interaction with NNN are more similar. These results provide new mechanistic insights into the mode of interaction of the substrates with the active site of cytochrome 2A13, in the presence of compound I, which is essential for α-hydroxylation.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Simulação de Dinâmica Molecular , Nicotina/metabolismo , Nitrosaminas/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Biocatálise , Domínio Catalítico , Simulação de Acoplamento Molecular
9.
Arch Biochem Biophys ; 679: 108216, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31801692

RESUMO

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, ß-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Ácidos Graxos/metabolismo , Álcoois Graxos/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Citocromos b5/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Conformação Proteica
10.
Chem Res Toxicol ; 32(12): 2488-2498, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31799839

RESUMO

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.


Assuntos
Amidas/toxicidade , Hidrocarboneto de Aril Hidroxilases/metabolismo , Furanos/toxicidade , Ativação Metabólica , Amidas/síntese química , Amidas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/química , Domínio Catalítico , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/metabolismo , Inibidores das Enzimas do Citocromo P-450/toxicidade , Furanos/síntese química , Furanos/metabolismo , Células Hep G2 , Humanos , Hidroxilação , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Coelhos , Relação Estrutura-Atividade , Terpenos/química , Terpenos/toxicidade
11.
Chem Res Toxicol ; 32(12): 2466-2478, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31730751

RESUMO

Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4- and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 µM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 0.13 µM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω-1)-hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Compostos de Benzalcônio/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Desinfetantes/metabolismo , Amidinas/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/química , Compostos de Benzalcônio/química , Isótopos de Carbono/química , Citocromo P-450 CYP2D6/química , Inibidores do Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Feminino , Humanos , Hidroxilação/efeitos dos fármacos , Cinética , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Oxirredução , Quinidina/farmacologia
12.
Molecules ; 24(23)2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31775347

RESUMO

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%-120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug-drug interactions.


Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450/farmacologia , Família 2 do Citocromo P450/antagonistas & inibidores , Fígado/efeitos dos fármacos , Ácido Salicílico/farmacologia , Esteroide 16-alfa-Hidroxilase/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/química , Catálise , Cromatografia Líquida de Alta Pressão , Inibidores das Enzimas do Citocromo P-450/química , Família 2 do Citocromo P450/química , Desenvolvimento de Medicamentos , Humanos , Fígado/enzimologia , Ratos , Ácido Salicílico/química , Esteroide 16-alfa-Hidroxilase/química
13.
J Ind Microbiol Biotechnol ; 46(5): 635-647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30790119

RESUMO

Cholesterol oxidase, steroid C27 monooxygenase and 3-ketosteroid-Δ1-dehydrogenase are key enzymes involved in microbial catabolism of sterols. Here, three isoenzymes of steroid C27 monooxygenase were firstly characterized from Mycobacterium neoaurum as the key enzyme in sterol C27-hydroxylation. Among these three isoenzymes, steroid C27 monooxygenase 2 exhibits the strongest function in sterol catabolism. To improve androst-1,4-diene-3,17-dione production, cholesterol oxidase, steroid C27 monooxygenase 2 and 3-ketosteroid-Δ1-dehydrogenase were coexpressed to strengthen the metabolic flux to androst-1,4-diene-3,17-dione, and 3-ketosteroid 9α-hydroxylase, which catalyzes the androst-1,4-diene-3,17-dione catabolism, was disrupted to block the androst-1,4-diene-3,17-dione degradation pathway in M. neoaurum JC-12. Finally, the recombinant strain JC-12S2-choM-ksdd/ΔkshA produced 20.1 g/L androst-1,4-diene-3,17-dione, which is the highest reported production with sterols as substrate. Therefore, this work is hopes to pave the way for efficient androst-1,4-diene-3,17-dione production through metabolic engineering.


Assuntos
Androstadienos/química , Isoenzimas/metabolismo , Micobactérias não Tuberculosas/metabolismo , Fitosteróis/metabolismo , Esteróis/química , Hidrocarboneto de Aril Hidroxilases/química , Microbiologia Industrial , Engenharia Metabólica , Metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredutases/química , Plasmídeos/metabolismo , Polienos/metabolismo , Esteroide Hidroxilases/química
14.
Chem Res Toxicol ; 32(3): 484-492, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30701961

RESUMO

Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with H2O2 and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S-) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Glutamina/farmacologia , Heme/metabolismo , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Coelhos
15.
Xenobiotica ; 49(11): 1323-1331, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30596462

RESUMO

1. Propafenone, an antiarrhythmic drug, is a typical human cytochrome P450 (P450) 2D6 substrate used in preclinical studies. Here, propafenone oxidation by mammalian liver microsomes was investigated in vitro. 2. Liver microsomes from humans and marmosets preferentially mediated propafenone 5-hydroxylation, minipig, rat and mouse livers primarily mediated 4'-hydroxylation, but cynomolgus monkey and dog liver microsomes differently mediated N-despropylation. 3. Quinine, ketoconazole or anti-P450 2D antibodies suppressed propafenone 4'/5-hydroxylation in human and rat liver microsomes. Pretreatments with ß-naphthoflavone or dexamethasone increased N-despropylation in rat livers. 4. Recombinant rat P450 2D2 efficiently catalysed propafenone 4'-hydroxylation in a substrate inhibition manner, comparable to rat liver microsomes, while human P450 2D6 displayed propafenone 5-hydroxylation. Human and rat P450 1A, 2C and 3A enzymes mediated propafenone N-despropylation with high capacities. 5. Carbon-4' of propafenone docked favourably into the active site of P450 2D2 based on an in silico model; in contrast, carbon-5 of propafenone docked into human P450 2D6. 6. These results suggest that the major roles of individual P450 2D enzymes in regioselective hydroxylations of propafenone differ between human and rat livers, while the minor roles of P450 1A, 2C and 3A enzymes for propafenone N-despropylation are similar in livers of both species.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Microssomos Hepáticos/metabolismo , Propafenona/farmacocinética , Adulto , Idoso , Animais , Antiarrítmicos/química , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacocinética , Hidrocarboneto de Aril Hidroxilases/química , Callithrix , Citocromo P-450 CYP2D6/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Cães , Feminino , Humanos , Hidroxilação , Macaca fascicularis , Masculino , Camundongos Endogâmicos , Microssomos Hepáticos/efeitos dos fármacos , Pessoa de Meia-Idade , Propafenona/química , Propafenona/metabolismo , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura
16.
J Biol Chem ; 293(29): 11433-11446, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29858244

RESUMO

Cytochrome P450 4B1 (4B1) functions in both xenobiotic and endobiotic metabolism. An ester linkage between Glu-310 in 4B1 and the 5-methyl group of heme facilitates preferential hydroxylation of terminal (ω) methyl groups of hydrocarbons (HCs) and fatty acids compared with ω-1 sites bearing weaker C-H bonds. This preference is retained albeit diminished 4-fold for the E310A mutant, but the reason for this is unclear. Here, a crystal structure of the E310A-octane complex disclosed that noncovalent interactions maintain heme deformation in the absence of the ester linkage. Consistent with the lower symmetry of the heme, resonance Raman (RR) spectroscopy revealed large enhancements of RR peaks for high-spin HC complexes of 4B1 and the E310A mutant relative to P450 3A4. Whereas these enhancements were diminished in RR spectra of a low-spin 4B1-N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine complex, a crystal structure indicated that this inhibitor does not alter heme ruffling. RR spectra of Fe2+-CO HC complexes revealed larger effects of HC length in E310A than in 4B1, suggesting that reduced rigidity probably underlies increased E310A-catalyzed (ω-1)-hydroxylation. Diminished effects of the HC on the position of the Fe-CO stretching mode in 4B1 suggested that the ester linkage limits substrate access to the CO. Heme ruffling probably facilitates autocatalytic ester formation by reducing inhibitory coordination of Glu-310 with the heme iron. This also positions the 5-methyl for a reaction with the proposed glutamyl radical intermediate and potentially enhances oxo-ferryl intermediate reactivity for generation of the glutamyl radical to initiate ester bond formation and ω-hydroxylation.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Heme/química , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Heme/metabolismo , Hidroxilação , Modelos Moleculares , Oxirredução , Coelhos , Análise Espectral Raman , Estereoisomerismo , Especificidade por Substrato
19.
J Mol Model ; 24(3): 67, 2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29476269

RESUMO

The interactions of the drugs amlodipine and paroxetine, which are prescribed respectively for treatment of hypertension and depression, with the metabolizing enzyme cytochrome CYP2B4 as the drug target, have been studied by molecular dynamics (MD) simulation. Poly ethylene glycol was used to control the drugs' interactions with each other and with the target CYP2B4. Thirteen simulation systems were carefully designed, and the results obtained from MD simulations indicated that amlodipine in the PEGylated form prescribed with paroxetine in the nonPEGylated form promotes higher cytochrome stability and causes fewer fluctuations as the drugs approach the target CYP2B4 and interact with it. The simulation results led us to hypothesize that the combination of the drugs with a specific drug ratio, as proposed in this work, manifests more effective diffusivity and less instability while metabolizing with enzyme CYP2B4. Also, the active residues in the CYP2B4 enzyme that interact with the drugs were determined by MD simulation, which were consistent with the reported experimental results. Graphical Abstract Efficient drug-enzyme interactions, as a result of PEGylation.


Assuntos
Anlodipino/química , Antidepressivos de Segunda Geração/química , Anti-Hipertensivos/química , Hidrocarboneto de Aril Hidroxilases/química , Paroxetina/química , Anlodipino/metabolismo , Antidepressivos de Segunda Geração/metabolismo , Anti-Hipertensivos/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/química , Família 2 do Citocromo P450/metabolismo , Difusão , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Paroxetina/metabolismo
20.
Drug Metab Pharmacokinet ; 33(1): 82-89, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29342418

RESUMO

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.


Assuntos
Alelos , Hidrocarboneto de Aril Hidroxilases/genética , Cumarínicos/metabolismo , Variação Genética/genética , Imidazóis/metabolismo , Nicotina/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Células HEK293 , Humanos , Hidroxilação , Oxirredução , Estrutura Secundária de Proteína
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