RESUMO
The cytochrome P450 2C19 (CYP2C19) enzyme plays an important role in the metabolism of many commonly used drugs. Relatively little is known about CYP2C19 inhibitors, including compounds of natural origin, which could inhibit CYP2C19, potentially causing clinically relevant metabolism-based drug interactions. We evaluated a series (N = 49) of structurally related plant isoquinoline alkaloids for their abilities to interact with CYP2C19 enzyme using in vitro and in silico methods. We examined several common active alkaloids found in herbal products such as apomorphine, berberine, noscapine, and papaverine, as well as the previously identified mechanism-based inactivators bulbocapnine, canadine, and protopine. The IC50 values of the alkaloids ranged from 0.11 to 210 µM, and 42 of the alkaloids were confirmed to be time-dependent inhibitors of CYP2C19. Molecular docking and three-dimensional quantitative structure-activity relationship analysis revealed key interactions of the potent inhibitors with the enzyme active site. We constructed a comparative molecular field analysis model that was able to predict the inhibitory potency of a series of independent test molecules. This study revealed that many of these isoquinoline alkaloids do have the potential to cause clinically relevant drug interactions. These results highlight the need for studying more profoundly the potential interactions between drugs and herbal products. When further refined, in silico methods can be useful in the high-throughput prediction of P450 inhibitory potential of pharmaceutical compounds.
Assuntos
Alcaloides/química , Simulação por Computador , Inibidores do Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP2C19/metabolismo , Isoquinolinas/química , Extratos Vegetais/química , Alcaloides/farmacologia , Inibidores do Citocromo P-450 CYP2C19/farmacologia , Humanos , Isoquinolinas/farmacologia , Extratos Vegetais/farmacologia , Relação Quantitativa Estrutura-Atividade , Fatores de TempoRESUMO
Several in vitro criteria were used to assess whether three methylenedioxyphenyl (MDP) compounds, the isoquinoline alkaloids bulbocapnine, canadine, and protopine, are mechanism-based inactivators of CYP2C19. The recently reported fluorometric CYP2C19 progress curve analysis approach was applied first to determine whether these alkaloids demonstrate time-dependent inhibition. In this experiment, bulbocapnine, canadine, and protopine displayed time dependence and saturation in their inactivation kinetics with K(I) and k(inact) values of 72.4 ± 14.7 µM and 0.38 ± 0.036 min(-1), 2.1 ± 0.63 µM and 0.18 ± 0.015 min(-1), and 7.1 ± 2.3 µM and 0.24 ± 0.021 min(-1), respectively. Additional studies were performed to determine whether other specific criteria for mechanism-based inactivation were fulfilled: NADPH dependence, irreversibility, and involvement of a catalytic step in the enzyme inactivation. CYP2C19 activity was not significantly restored by dialysis when it had been inactivated by the alkaloids in the presence of a NADPH-regenerating system, and a metabolic-intermediate complex-associated increase in absorbance at approximately 455 nm was observed. In conclusion, the CYP2C19 progress curve analysis method revealed time-dependent inhibition by these alkaloids, and additional experiments confirmed its quasi-irreversible nature. This study revealed that the CYP2C19 progress curve analysis method is useful for identifying novel mechanism-based inactivators and yields a wealth of information in one run. The alkaloids bulbocapnine, canadine, and protopine, present in herbal medicines, are new mechanism-based inactivators and the first MDP compounds exhibiting quasi-irreversible inactivation of CYP2C19.
Assuntos
Aporfinas/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzofenantridinas/farmacologia , Alcaloides de Berberina/farmacologia , Berberina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Berberina/farmacologia , Citocromo P-450 CYP2C19 , HumanosRESUMO
Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using synthesized fluorescein benzyl ester and fluorescein benzyl ether along with commercially available fluorescein as intermediate standards. Furthermore, we demonstrated the use of DBF in a kinetic assay as a progress curve analysis for straightforward determination of whether a compound is a time-dependent inactivator of CYP2C19. The recombinant human CYP2C19 inactivation kinetics of isoniazid, ticlopidine, and tranylcypromine were evaluated, and their key kinetic parameters were measured from the same experiment. The known mechanism-based inactivators, isoniazid and ticlopidine, exhibited clear time-dependent inactivation with K(I) and k(inact) values of 250.5 ± 34 µM and 0.137 ± 0.006 min(-1) and 1.96 ± 0.5 µM and 0.135 ± 0.009 min(-1), respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biocatálise , Citocromo P-450 CYP2C19 , Interações Medicamentosas , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Isoniazida/farmacologia , Cinética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Ticlopidina/farmacologia , Tranilcipromina/farmacologiaRESUMO
The human CYP2A6 enzyme metabolises several xenobiotics including nicotine, the addictive component in tobacco. Reduced activity of CYP2A6, either for genetic reasons or by administering inhibitors of CYP2A6, reduces tobacco smoking. The aim was to design novel inhibitors of CYP2A6 using 3D-QSAR analysis combined with virtual screening. A 3D-QSAR model was utilised to identify the most important features of the inhibitors, and this knowledge was used to design inhibitors for CYP2A6. Chemical database screening yielded several potent inhibitor candidates such as alkylamine derivatives (compound no. 5, IC(50)=0.1 µM) and 1-benzothiophene-3-carbaldehyde that can be used as lead compounds in the development of drugs for smoking reduction therapy.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2A6 , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Cinética , Modelos Moleculares , Relação Quantitativa Estrutura-AtividadeRESUMO
Because of the increasing number of immunocompromised patients and due to problems with antifungal treatment, especially with the most widely used antifungals, azoles, there is an urgent need for new, potent and safe antifungals with fewer cytochrome P450 (CYP)-mediated interactions with other drugs. In the present study, 54 novel non-azole molecules were selected with the help of molecular modelling and virtual molecule database screening to identify new fungistatic or fungicidic compounds with functional groups that would produce reactive intermediates killing the yeast cells. Database screening and selection of tested compounds were based on the construction of two pharmacophores and docking hits to the active site of the CYP51 homology model. Inhibition potency of the compounds was tested against Saccharomyces cerevisiae and/or Candida albicans. Two new structured compounds, 2-({4-[(2-cyanoethyl)(methyl) amino]benzylidene} amino)-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile and 2-[([1,1'-biphenyl]-4-ylmethylene)amino]-5-(3,4-dimethoxyphenyl)-4-methylthiophene-3-carbonitrile were discovered to have promising antifungal properties based on bioassays. Inhibition screen of human hepatic CYP enzymes revealed that these two compounds did not inhibit potently five human recombinant CYP enzymes. The results of this study indicate that the functional groups of the two compounds may produce reactive intermediates when located at the active site of CYP51.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Nitrilas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Antifúngicos/química , Candida albicans/enzimologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Fígado/enzimologia , Modelos Moleculares , Nitrilas/química , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/metabolismo , Interface Usuário-ComputadorRESUMO
The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n=36) consist of compounds isolated from the Papaveraceae family (n=20), synthetic analogs (n=15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC50 < 10 µM) and 15/36 inhibiting CYP3A4 potently (IC50 < 1 µM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.