RESUMEN
Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 µM and 0.62 min-1, respectively. The calculated kobs at 10 µM was 0.29 min-1, above the 0.02 min-1 risk level. After incubation with NOT at 10 µM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.
Asunto(s)
Cumarinas/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Activación Metabólica , Apiaceae/química , Apiaceae/metabolismo , Cumarinas/química , Citocromo P-450 CYP2D6/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/metabolismoRESUMEN
The synthesis, the antioxidative properties and the lipoxygenase (LOX) and acetylcholinesterase (AChE) inhibition of a number of 4-hydroxy-chalcones diversely substituted as well as of a series of bis-chalcones ether derivatives are reported. The chalcones derivatives were readily produced using a Claisen-Schmidt condensation in a ultra sound bath in good yields. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity is experimentally determined by reversed-phase thin-layer chromatography method. Most of them are potent in vitro inhibitors of lipid peroxidation and of LOX. Compounds b2 and b3 were found to be the most potent LOX and AChE inhibitors among the tested derivatives with a significant anti-lipid peroxidation profile. The results led us to propose these enone derivatives as new multifunctional compounds against Alzheimer's disease. The results are discussed in terms of structural and physicochemical characteristics of the compounds. Moreover, the pharmacokinetic profile of these compounds was investigated using computational methods.
Asunto(s)
Chalconas/química , Chalconas/farmacología , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Animales , Antioxidantes/química , Antioxidantes/farmacología , Células CACO-2 , Línea Celular , Chalconas/síntesis química , Técnicas de Química Sintética , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Cromatografía en Capa Delgada , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Glutatión/química , Humanos , Peroxidación de Lípido/efectos de los fármacos , Inhibidores de la Lipooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa/química , Ratones , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.
Asunto(s)
Benzazepinas/química , Antagonistas de los Receptores Histamínicos H3/química , Receptores Histamínicos H3/química , Animales , Benzazepinas/síntesis química , Benzazepinas/farmacocinética , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Evaluación Preclínica de Medicamentos , Semivida , Antagonistas de los Receptores Histamínicos H3/síntesis química , Antagonistas de los Receptores Histamínicos H3/farmacocinética , Humanos , Microsomas Hepáticos/metabolismo , Unión Proteica , Ratas , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-ActividadRESUMEN
The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.
Asunto(s)
Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Simulación del Acoplamiento Molecular/métodos , Dominio Catalítico , Citocromo P-450 CYP2D6/química , Interacciones de Hierba-Droga , Medicina de Hierbas/métodos , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Medicina Tradicional China/métodos , Mutación , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Especificidad por SustratoRESUMEN
A novel series of malonamide derivatives was synthesized. These amides were shown to be potent and selective kappa opioid receptor agonists.
Asunto(s)
Química Farmacéutica/métodos , Malonatos/síntesis química , Malonatos/farmacología , Receptores Opioides kappa/antagonistas & inhibidores , Citocromo P-450 CYP2D6/química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Cinética , Malonatos/química , Modelos Químicos , Conformación MolecularRESUMEN
Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2D6 , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales/química , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/química , Indonesia , Medicina Tradicional , Extractos Vegetales/químicaRESUMEN
A series of fluorescent molecularly imprinted polymers has been prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. Such polymers would have the possibility to form the sensing element in a high-throughput assay for the prediction of CYP2D6 affinity. The imprinted polymers possessed binding-dependent fluorescence. They re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One polymer in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from a drug panel.
Asunto(s)
Materiales Biomiméticos/química , Citocromo P-450 CYP2D6/análisis , Citocromo P-450 CYP2D6/química , Colorantes Fluorescentes/química , Modelos Moleculares , Polímeros/química , Espectrometría de Fluorescencia/métodos , Sitios de Unión , Materiales Biomiméticos/análisis , Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/análisis , Preparaciones Farmacéuticas/química , Unión ProteicaRESUMEN
1. Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue. Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery. 2. In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)). Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters. Results were compared with data obtained in human liver microsomes (HLM). 3. Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max). K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold. V(max) differed up to 3-fold between matrices for the CYP forms investigated. However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM. The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged. 4. In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters. Furthermore, addition of antioxidant can in certain cases increase catalytic activity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Citocromo P-450 CYP2D6/química , Sistema Enzimático del Citocromo P-450/química , Evaluación Preclínica de Medicamentos/métodos , Microsomas Hepáticos/enzimología , Oxidorreductasas O-Demetilantes/química , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilasas/química , Antioxidantes/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Ácido Ascórbico/farmacología , Catálisis , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Depuradores de Radicales Libres/farmacología , Humanos , Cinética , Oxidorreductasas O-Demetilantes/metabolismo , Proteínas Recombinantes/química , Esteroide Hidroxilasas/metabolismo , Factores de TiempoRESUMEN
High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/química , Evaluación Preclínica de Medicamentos/métodos , Conteo por Cintilación/métodos , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Cinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Químicos , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidorreductasas O-Demetilantes/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen. In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6. An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems. Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase. The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301. Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol. Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme. These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase.