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2.
Pharmacol Biochem Behav ; 156: 30-38, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28373066

RESUMO

Benzphetamine is a Schedule III anorectic agent that is a prodrug for d-amphetamine and d-methamphetamine and may have utility as an "agonist" medication for cocaine use disorder treatment. This study evaluated the pharmacokinetic-pharmacodynamic profile of benzphetamine using a drug discrimination procedure in rhesus monkeys. The potency and time course of cocaine-like discriminative stimulus effects were compared for benzphetamine (10-18mg/kg, intramuscular (IM)) and d-amphetamine (0.032-0.32mg/kg, IM) in monkeys (n=3-4) trained to discriminate IM cocaine (0.32mg/kg) from saline in a two-key food-reinforced discrimination procedure. Parallel pharmacokinetic studies in the same monkeys determined plasma benzphetamine, d-methamphetamine and/or d-amphetamine levels for correlation with behavioral effects. d-Amphetamine produced dose-dependent, time-dependent, and full cocaine-like effects, i.e. ≥90% cocaine-appropriate responding, in all monkeys without altering response rates. The time course of d-amphetamine's cocaine-like discriminative stimulus effects correlated with plasma d-amphetamine levels. Benzphetamine was 180-fold less potent than d-amphetamine and produced full cocaine-like effects in only 2 of 4 monkeys while significantly decreasing response rates. Benzphetamine administration increased plasma d-methamphetamine (peak at 100min) and d-amphetamine (peak at 24h) levels, but the time course of behavioral effects did not correlate with increased levels of benzphetamine, d-methamphetamine or d-amphetamine. These results suggest that benzphetamine yields d-amphetamine and d-methamphetamine as active metabolites in rhesus monkeys, but generation of these metabolites is not sufficient to account for benzphetamine behavioral effects. The incomplete cocaine substitution profile and protracted d-amphetamine plasma levels suggest that benzphetamine may still warrant further evaluation as a candidate pharmacotherapy for cocaine use disorder treatment.


Assuntos
Benzfetamina/farmacologia , Dextroanfetamina/farmacologia , Metanfetamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Benzfetamina/farmacocinética , Dextroanfetamina/farmacocinética , Macaca mulatta , Masculino , Metanfetamina/farmacocinética
3.
J Neurosci ; 33(31): 12792-804, 2013 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-23904614

RESUMO

Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with memory impairments in rats. In human, prodynorphin (Pdyn) gene polymorphisms might be linked to cognitive function in the elderly. Moreover, elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease patients. However, the cellular and molecular processes affected by higher dynorphin levels during aging remain unknown. Using Pdyn(-/-) mice, we observed significant changes in the function and expression of Group 1 metabotropic glutamate receptor (mGluR). Compared with age-matched wild-type (WT) littermates, we found increased expression of mGluR1α and mGluR5 in the hippocampus and cortex of old, but not young, Pdyn(-/-) mice. Increased Group 1 mGluR expression in aged Pdyn(-/-) mice was associated with enhanced mGluR-mediated long-term depression, a form of synaptic plasticity. Notably, whereas aged WT mice developed spatial and recognition memory deficits, aged Pdyn(-/-) mice performed similarly as young mice. Pharmacological treatments with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, a positive modulator of mGlu5 receptors, or norbinaltorphimine, an antagonist for dynorphin-targeted κ-opioid receptor, rescued memory in old WT mice. Conversely, mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride impaired spatial memory of old Pdyn(-/-) mice. Intact cognition in aged Pdyn(-/-) mice paralleled with increased expression of Group 1 mGluR-related genes Homer 1a and Arc. Finally, aged Pdyn(-/-) mice displayed less anxiety-related behaviors than age-matched WT mice. Together, our results suggest that elevated Pdyn expression during normal aging reduces mGluR expression and signaling, which in turn impairs cognitive functions and increases anxiety.


Assuntos
Envelhecimento/fisiologia , Ansiedade/metabolismo , Encefalinas/deficiência , Regulação da Expressão Gênica/genética , Precursores de Proteínas/deficiência , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Ansiedade/tratamento farmacológico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Benzfetamina/análogos & derivados , Benzfetamina/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/genética , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia
4.
Biosens Bioelectron ; 26(9): 3914-9, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21497079

RESUMO

This work focuses on P450 biosensors based on multiwalled carbon nanotubes (MWCNT) and different cytochrome isoforms: 3A4, 2B4, 2C9. The proposed biosensors exhibit enhanced sensitivities and decreased detection limits thanks to carbon nanotubes. The MWCNT structuring improves the sensitivity from 5.1 to 20.5 nA/mM mm(2) in case of CYP2B4-mediated Benzphetamine detection, from 0.26 to 0.63 nA/µM mm(2) in case of CYP3A4-mediated Cyclophosphamide detection, and from 0.11 to 0.25 nA/µM mm(2) in case of CYP2C9-mediated Naproxen detection. By using MWCNT, the limit of detection was enhanced from 59 to 12 µM in case of Cyclophosphamide and from to 187 to 82 µM in case of Naproxen. This makes possible the drug detection in human serum within the pharmacological range. In the paper, a new mathematical model is also proposed to succeed in discriminating different drug contributions in a mixture containing both Cyclophosphamide and Dextromethorphan or combining Naproxen and Flurbiprofen. Data analysis shows variations in reduction peaks that are dependent on the drug ratio, and that are consistent with competitive kinetics of substrates. This new approach enables multiple drug detection and opens the way to possible applications in personalized therapy.


Assuntos
Benzfetamina/isolamento & purificação , Técnicas Biossensoriais , Ciclofosfamida/isolamento & purificação , Naproxeno/isolamento & purificação , Hidrocarboneto de Aril Hidroxilases/química , Benzfetamina/química , Ciclofosfamida/química , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP3A/química , Família 2 do Citocromo P450 , Humanos , Limite de Detecção , Nanotubos de Carbono/química , Naproxeno/química
5.
Biochim Biophys Acta ; 1814(1): 69-75, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20637316

RESUMO

Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O2) complex of CYP2B4, homogeneous O2 complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~-50°C in mixed solvents (60-70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~4 nm and ~3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Mutação , Oxigênio/metabolismo , Substituição de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/química , Benzfetamina/química , Benzfetamina/metabolismo , Domínio Catalítico , Dicroísmo Circular , Temperatura Baixa , Família 2 do Citocromo P450 , Heme/química , Heme/metabolismo , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Oxigênio/química , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Espectrofotometria , Especificidade por Substrato
6.
Biochemistry (Mosc) ; 75(7): 899-904, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20673214

RESUMO

It is shown that a stable nitroxyl radical, 4-cyano-2,2,6,6-tetramethylpiperidine-1-oxyl, forms a complex with cytochrome P4502B4 by analogy with the second type substrates by joining directly to pentacoordinate heme iron. The bound radical is inaccessible to water-soluble paramagnetic ions, which confirms its localization in a hydrophobic pocket near the heme. Benzphetamine and N,N-dimethylaniline, the first-type nonpolar substrates, induce conformational changes of the spin-labeled hemoprotein which are evidently accompanied by an increase in the volume of the pocket resulting in emergence of contact with aqueous phase, and the heme-bound spin label becomes accessible to water-soluble paramagnetics. In this case potassium ferricyanide broadens the spin-labeled cytochrome signal and, as a result, lowers the amplitudes of the spectral components. Similar changes were registered at non-micellar concentrations of nonionic detergent Emulgen 913, whose activating effect on hydroxylation reactions is associated, as we showed previously, with its presence in the CYP2B4 active site simultaneously with substrates.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Domínio Catalítico , Microssomos Hepáticos/enzimologia , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzfetamina/química , Benzfetamina/metabolismo , Família 2 do Citocromo P450 , Heme/química , Heme/metabolismo , Ligantes , Microssomos Hepáticos/química , Ligação Proteica , Conformação Proteica , Coelhos , Especificidade por Substrato
7.
J Vet Pharmacol Ther ; 33(3): 295-303, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20557447

RESUMO

The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C- and 3A-mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin-containing monooxygenases (methimazole S-oxidation), and uridindiphosphoglucuronyltransferases (1-naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S-transferase (GST)-dependent conjugation of 1-chloro-2,4-dinitrobenzene; besides, the GST-mediated conjugation of ethacrinic acid (GSTpi) or cumene hydroperoxide (GSTalpha) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4-dichloronitrobenzene, a protein cross reacting with anti-rat GSTmu antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Fatores Etários , Animais , Benzfetamina/metabolismo , Biotransformação , Western Blotting , Bovinos , Clorfeniramina/metabolismo , Dieta/veterinária , Duodeno/enzimologia , Eletroforese em Gel de Poliacrilamida , Etilmorfina/metabolismo , Glutationa Transferase/metabolismo , Masculino , Microssomos/enzimologia
8.
Mol Pharmacol ; 76(5): 1011-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19720728

RESUMO

We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.


Assuntos
Acetileno/análogos & derivados , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Acetileno/farmacologia , Benzfetamina/farmacologia , Catálise/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Família 2 do Citocromo P450 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estereoisomerismo , Especificidade por Substrato/efeitos dos fármacos
9.
J Mol Graph Model ; 28(2): 170-6, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19596602

RESUMO

The human cytochrome P450 2W1 (CYP2W1) was categorized into the so-called "orphan" CYPs because of its unknown enzymatic function. However, recent studies showed that the recombinant CYP2W1 exhibited broad catalytic activity towards several chemicals. Furthermore, this enzyme was selectively expressed in some forms of cancers, whereas a very low expression was found in human normal issues. These render CYP2W1 as a potential drug target for cancer therapy. At present, however, little information is available on the active site topology and the substrate binding modes of CYP2W1. In this study, the three-dimensional model of CYP2W1 was constructed using the homology modeling method. Two known substrates, benzphetamine and indole, were then docked into the active site, and refined by molecular dynamics simulations. The interaction energy between the substrates and the enzyme was calculated and analyzed by using the MM-GBSA method. The results indicated that the constructed CYP2W1 model can account for the regioselectivity of this enzyme towards the known substrates and van der Waals interactions were the driving force for the substrate binding. Several key residues were identified to be responsible for the binding of indole and benzphetamine with CYP2W1. These findings provide useful information for the detailed characterization of the biological roles of CYP2W1 and structure-based drug design of this enzyme.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Benzfetamina/química , Benzfetamina/metabolismo , Família 2 do Citocromo P450 , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
10.
Bioorg Med Chem ; 17(6): 2452-60, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19243954

RESUMO

A series of naphthylisopropylamine and N-benzyl-4-methylthioamphetamine derivatives were evaluated as monoamine oxidase inhibitors. Their potencies were compared with those of a series of amphetamine derivatives, to test if the increase of electron richness of the aromatic ring and overall size of the molecule might improve their potency as enzyme inhibitors. Molecular dockings were performed to gain insight regarding the binding mode of these inhibitors and rationalize their different potencies. In the case of naphthylisopropylamine derivatives, the increased electron-donating capacity and size of the aromatic moiety resulting from replacement of the phenyl ring of amphetamine derivatives by a naphthalene system resulted in more potent compounds. In the other case, extension of the arylisopropylamine molecule by N-benzylation of the amino group led to a decrease in potency as monoamine oxidase inhibitors.


Assuntos
Benzfetamina/análogos & derivados , Inibidores da Monoaminoxidase/farmacologia , Naftalenos/farmacologia , Propilaminas/farmacologia , Animais , Benzfetamina/química , Benzfetamina/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Inibidores da Monoaminoxidase/química , Naftalenos/química , Propilaminas/química , Ratos
11.
J Am Chem Soc ; 131(8): 2971-6, 2009 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19209859

RESUMO

Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized benzphetamine to norbenzphetamine in high yield in bulk studies. In direct kinetic studies of benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (K(bind)) for each. The first-order oxidation rate constants (k(ox)) were comparable for Compounds I of CYP2B4, the E301Q mutant, and the T302A mutant, whereas the k(ox) for Compound I of the F429H mutant was reduced by a factor of 2. CYP119 Compound I, studied for comparison purposes, reacted with benzphetamine with a binding constant that was nearly an order of magnitude smaller than that of CYP2B4 but a rate constant that was similar. Substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions, and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Benzfetamina/química , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzfetamina/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Cinética , Modelos Moleculares , Oxirredução , Processos Fotoquímicos , Fotólise , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta
12.
Drug Metab Dispos ; 37(4): 745-52, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19144770

RESUMO

Phencyclidine (PCP) is a mechanism-based inactivator of cytochrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphetamine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-octylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k(1) = 5.0 s(-1), A(1) = 30%; k(2) = 0.02 s(-1), A(2) = 70%, where A(2) indicates the fractional magnitude of the second phase) compared with the native enzyme (k(1) = 8.0 s(-1), A(1) = 58%; k(2) = 0.3 s(-1), A(2) = 42%). Analysis of benzphetamine metabolism by the inactivated protein using liquid chromatography/electrospray ionization/mass spectrometry showed that the rates of formation of nor-benzphetamine and hydroxylated nor-benzphetamine were decreased by 75 and 69%, respectively, whereas the rates of formation for amphetamine, hydroxybenzphetamine, and methamphetamine showed slight but statistically insignificant decreases after the inactivation. The rate of reduction of P450 2B6 by NADPH and reductase was decreased by 6-fold as a result of the modification by PCP. In addition, the extent of uncoupling of NADPH oxidation from product formation, a process leading to futile production of H(2)O(2), increased significantly during the metabolism of ethylbenzene as a result of the inactivation.


Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Fenciclidina/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzfetamina/farmacocinética , Catálise , Cromatografia Líquida , Citocromo P-450 CYP2B6 , Transporte de Elétrons , Oxirredutases N-Desmetilantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
13.
J Biol Chem ; 284(17): 11374-84, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19171935

RESUMO

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.


Assuntos
NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/fisiologia , Animais , Benzfetamina/metabolismo , Elétrons , Mononucleotídeo de Flavina/química , Flavinas/química , Deleção de Genes , Cinética , Conformação Molecular , Mutagênese , Mutação , Oxirredução , Conformação Proteica , Estrutura Terciária de Proteína , Ratos
14.
Mol Pharmacol ; 74(4): 990-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18621926

RESUMO

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-alpha-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Mutação , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Alcinos , Benzoxazinas/química , Benzoxazinas/metabolismo , Benzfetamina/metabolismo , Sítios de Ligação , Catálise , Ciclopropanos , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2B6 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/isolamento & purificação , Citocromos b5/metabolismo , Escherichia coli/genética , Etinilestradiol/análogos & derivados , Etinilestradiol/metabolismo , Humanos , Cinética , Estrutura Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxidantes/metabolismo , Ligação Proteica , Espectrofotometria Ultravioleta , terc-Butil Hidroperóxido/metabolismo
15.
Biochemistry ; 47(12): 3950-63, 2008 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-18311926

RESUMO

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Benzfetamina/metabolismo , Hidroxitolueno Butilado/metabolismo , Animais , Família 2 do Citocromo P450 , Citocromos b5/metabolismo , Compostos Férricos/metabolismo , Manganês/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Coelhos , Análise Espectral Raman
16.
J Mol Biol ; 377(1): 232-45, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18241887

RESUMO

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (Delta Delta G) of approximately 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a Delta Delta G of approximately 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a Delta Delta G of approximately 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Benzfetamina/química , Benzfetamina/farmacologia , Sítios de Ligação , Calorimetria , Família 2 do Citocromo P450 , Inibidores Enzimáticos/química , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica , Água/química
19.
J Biol Chem ; 282(41): 29766-76, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17693640

RESUMO

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios

Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Citocromos b5/fisiologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Benzfetamina/análogos & derivados , Benzfetamina/química , Sítios de Ligação , Ligação Competitiva , Cromatografia Líquida , Cicloexanóis/química , Família 2 do Citocromo P450 , Citocromos b5/química , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , NADP/química , Coelhos , Ratos , Especificidade por Substrato
20.
Chirality ; 19(8): 647-53, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17568428

RESUMO

The HPLC enantiomeric separation of N-benzyl-alpha-methyl-benzylamine, phenylalaninol, tryptophanol, 2 (diphenylhydroxymethyl)pyrrolidine, and isoproterenol was accomplished in the normal-phase mode using two polysaccharide-derived chiral stationary phases (CSPs) and various n-hexane/2-propanol mobile phases with acidic (TFA) or basic (DEA) additive. The compounds were separated without any derivatization and separation factor range between 2.09 and 1.09 with resolution factor 3.4 and 0.4, respectively. The best separation of the enantiomers of the amine was achieved on amylose tris (3, 5-dimethylphenylcarbamate) CSP with TFA additive in the mobile phase; in acidic conditions, instead, the best enantioseparation of the aminoalcohols was achieved on cellulose tris (3, 5-dimethylphenilcarbamate). A long equilibration time of the CSP when switching from an undoped mobile phase to a doped one is required to obtain reproducible results.


Assuntos
Aminas/isolamento & purificação , Amino Álcoois/isolamento & purificação , Ácidos , Aminas/química , Amino Álcoois/química , Benzfetamina/análogos & derivados , Benzfetamina/química , Benzfetamina/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Isoproterenol/química , Isoproterenol/isolamento & purificação , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/isolamento & purificação , Polissacarídeos , Pirrolidinas/química , Pirrolidinas/isolamento & purificação , Estereoisomerismo , Triptofano/análogos & derivados , Triptofano/química , Triptofano/isolamento & purificação
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