RESUMEN
Many breast tumors are hormone-dependent, and estrogens, especially estradiol (E2), have a pivotal role in their growth and development. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is a key enzyme in the biosynthesis of female sex steroids, catalyzing the NADPH-dependent reduction of estrone into biologically active estradiol. In this study, a library of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-one based compounds was synthesized, and the biological activities against 17beta-HSD1 in a cell-free and in a cell-based assay were evaluated. Several thieno[2,3-d]pyrimidin-4(3H)-one based compounds, at 0.1 and 1 muM test concentrations, were found to be potent 17beta-HSD1 inhibitors. For example, 4-(3-hydroxyphenylthio)-1,2,7,8,9,10,11,13-octahydro-13-oxo-[1]benzothieno[2',3':4,5]-pyrimido[1,2-a]azepine-3-carboxaldehyde (7f) is one of the most potent nonsteroidal 17beta-HSD1 inhibitors reported to date with 94% inhibition of the recombinant enzyme at 0.1 muM test concentration. Importantly, the majority of these compounds exhibited excellent selectivity over the oxidative isoform 17beta-HSD2 and lacked estrogenic effects in an estrogen receptor (ER) binding assay.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Azepinas/síntesis química , Pirimidinonas/síntesis química , Tiofenos/síntesis química , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Azepinas/química , Azepinas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Estradiol Deshidrogenasas/antagonistas & inhibidores , Estradiol Deshidrogenasas/metabolismo , Receptor alfa de Estrógeno/química , Receptor beta de Estrógeno/química , Estrógenos/síntesis química , Estrógenos/química , Estrógenos/farmacología , Femenino , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Modelos Moleculares , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/enzimología , Pirimidinonas/química , Pirimidinonas/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
The 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) enzyme regulates the conversion of estrone (E1) to the biologically active estradiol (E2). Due to its role as a key enzyme in female hormone production, it has emerged as an attractive drug target for inhibitor development in relation to hormone-dependent breast cancer. Herein, we report four pharmacophore models of 17beta-HSD1 based on a crystal structure, a relaxed crystal structure, a library of 17beta-HSD1 inhibitors and on a docked complex of 17betaHSD1 enzyme and a potent inhibitor. The models were used in screening two databases, which produced novel compounds to be used as leads in our drug design project. The results were validated by docking the compounds to the active site of the 17beta-HSD1 enzyme. With the help of our 3D-QSAR model, these results will be used to develop new inhibitors of 17beta-HSD1 as drug candidates.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Inhibidores Enzimáticos/análisis , Enlace de Hidrógeno , Relación Estructura-Actividad Cuantitativa , Reproducibilidad de los ResultadosRESUMEN
Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.
Asunto(s)
Aromatasa/metabolismo , Cumestrol/metabolismo , Lignanos/metabolismo , Modelos Moleculares , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Andrógenos/química , Andrógenos/metabolismo , Benzoflavonas/química , Benzoflavonas/metabolismo , Dominio Catalítico , Simulación por Computador , Cumestrol/química , Estrona/química , Estrona/metabolismo , Hemo/química , Lignanos/química , Fitoestrógenos/metabolismoRESUMEN
The 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) enzyme plays a crucial role in female hormonal regulation by catalysing the NADPH-dependent reduction of the less potent estrone E1 into the biologically active estradiol E2. Because 17beta-HSD1 is a key enzyme in E2 biosynthesis, it has emerged as an attractive drug target for inhibitor development. Herein we report the plausible binding modes and a 3D QSAR model of 17beta-HSD1 inhibitors based on a (di)cycloalkenothieno[2,3-d]pyrimidin-4(3H)-one core. Two generated enzyme complexes with potent inhibitors were subjected to molecular dynamics simulation to mimic the dynamic process of inhibitor binding. A set of 17beta-HSD1 inhibitors based on the thieno[2,3-d]pyrimidin-4(3H)-one core were docked into the resulting active site, and a CoMFA model employing the most extensive training set to date was generated. The model was validated with an external test set. Active site residues involved in inhibitor binding and CoMFA fields for steric and electrostatic interactions were identified. The model will be used to guide structural modifications of 17beta-HSD1 inhibitors based on a thieno[2,3-d]pyrimidin-4(3H)-one core in order to improve the biological activity as well as in the design of novel 17beta-HSD1 inhibitors.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Neoplasias de la Mama/tratamiento farmacológico , Simulación por Computador , Inhibidores Enzimáticos/uso terapéutico , Pirimidinonas/uso terapéutico , Relación Estructura-Actividad Cuantitativa , Tiofenos/uso terapéutico , Algoritmos , Sitios de Unión , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Catálisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ligandos , Conformación Molecular , NADP/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacología , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
Aromatase (CYP450(arom), CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme-substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.
Asunto(s)
Aromatasa/química , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Aromatasa/metabolismo , Diseño de Fármacos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Conejos , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The steryl ferulate contents of rye and wheat grains and their milling fractions were analyzed using a reversed-phase high-performance liquid chromatographic (HPLC) method. HPLC-mass spectrometry was used for identification. In addition, steryl ferulates of some selected milling byproducts were determined. The total steryl ferulate contents of rye and wheat grains were 6.0 and 6.3 mg/100 g, respectively. Uneven distribution of steryl ferulates in the grains led to considerable differences in the milling products; their steryl ferulate contents ranged from trace amounts in flours with low ash content to 20 and 34 mg/100 g in rye and wheat brans, respectively. Campestanyl ferulate and sitostanyl ferulate were the main components, followed by campesteryl ferulate and sitosteryl ferulate, whereas sitosterol was the main component in total sterols. Among the other samples, a byproduct of rice milling (pearling dust) was the best source of steryl ferulates, its total steryl ferulate content being 119 mg/100 g, whereas no measurable amounts of steryl ferulates were measured in oat bran or pearling dust of barley. The results indicated that rye and wheat and especially their bran fractions are comparable to corn as steryl ferulate sources.