RESUMEN
While searching for novel nonsteroidal inhibitors of human and rat prostatic 5alpha-reductases, we found a new series of indoline and aniline derivatives that showed potent inhibitory activities for both enzymes. Among them, 3-chloro-4-¿[1-(4-phenoxybenzyl)indolin-5-yl]oxylbenzoic acid (2e, YM-36117) showed a more potent inhibitory activity for the human enzyme than ONO-3805 with an IC50 value of 5.3 nM and a reduced rat prostatic dihydrotestosterone (DHT) concentration by oral administration. The synthesis and the structure-activity relationships of these indoline and aniline derivatives are presented.
Asunto(s)
Inhibidores de 5-alfa-Reductasa , Compuestos de Anilina/síntesis química , Inhibidores Enzimáticos/síntesis química , Indoles/síntesis química , Compuestos de Anilina/farmacología , Animales , Fenómenos Químicos , Química Física , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/farmacología , Masculino , Próstata/efectos de los fármacos , Próstata/enzimología , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
In a search for novel nonsteroidal inhibitors of human prostatic 5alpha-reductase, we found a new series of indole derivatives that showed potent inhibitory activities for the human enzyme. Among them, 4-[(1-benzyl-1H-indol-5-yl)oxyl-3-chlorobenzoic acid (2d, YM-32906) showed more potent inhibitory activity than finasteride with an IC50 value of 0.44 nM. 3-Chloro-4-[[1-(4-phenoxybenzyl)-1H-indol-5-yl]oxy]benzoic acid (2m) showed inhibitory activities for both human and rat prostatic 5alpha-reductase with IC50 values of 2.1 and 73 nM, respectively. The synthesis and structure-activity relationships of these indole derivatives are presented.
Asunto(s)
Inhibidores de 5-alfa-Reductasa , Inhibidores Enzimáticos/síntesis química , Indoles/síntesis química , Indoles/farmacología , Animales , Fenómenos Químicos , Química Física , Inhibidores Enzimáticos/química , Humanos , Concentración de Iones de Hidrógeno , Indoles/química , Masculino , Próstata/enzimología , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
In a search for novel nonsteroidal inhibitors of human prostatic 5 alpha-reductase, we found a new series of phenoxybenzoic acid derivatives to be potent human prostatic 5 alpha-reductase inhibitors. Among them, 4-(biphenyl-4-yloxy)benzoic acid derivatives (2n, YM-31758), 2o and 2s showed more potent inhibitory activities than finasteride with IC50 values of 0.87, 0.67 and 0.56 nM, respectively. The optimized structures for the phenoxybenzoic acid derivatives 2d-2i were calculated by molecular modeling analysis, and the favorable distance between the carbon of the carboxyl group and the centroid of the phenyl group (benzene ring C) was found to be in the 9-11 A range.
Asunto(s)
Inhibidores de 5-alfa-Reductasa , Benzoatos/síntesis química , Inhibidores Enzimáticos/síntesis química , Animales , Benzoatos/química , Benzoatos/farmacología , Células Cultivadas , Fenómenos Químicos , Química Física , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fibroblastos , Humanos , Técnicas In Vitro , Masculino , Próstata/efectos de los fármacos , Próstata/enzimología , Ratas , Ratas WistarRESUMEN
A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.
Asunto(s)
Oxidorreductasas de Alcohol/aislamiento & purificación , Oxidorreductasas de Alcohol/metabolismo , Pulmón/enzimología , Alcohol Deshidrogenasa/metabolismo , Oxidorreductasas de Alcohol/química , Aldehído Deshidrogenasa/metabolismo , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Inmunodifusión , Inmunohistoquímica , Cinética , Pulmón/citología , Sustancias Macromoleculares , Peso Molecular , NADP/metabolismo , Especificidad por Sustrato , PorcinosRESUMEN
The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing Ka values of 2-14 microM which were lower than the values of 21-125 microM for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the Ka values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both Vmax and [S]0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 microM. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Ácidos Grasos no Esterificados/farmacología , Ácidos Grasos Insaturados/farmacología , Pulmón/enzimología , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Detergentes/farmacología , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , Relación Estructura-Actividad , Especificidad por Sustrato , PorcinosRESUMEN
The kinetics of the NAD(P)(+)-linked aldehyde dismutation by pulmonary carbonyl reductase of guinea pig were studied using a highly hydrated substrate, chloral hydrate (CH). The enzyme irreversibly converted the substrate into trichloroacetic acid (TCA) and trichloroethanol (TCE) in the presence of the reduced or oxidized cofactors, of which NAD(P)+ gave a higher reaction rate than did NAD(P)H, and the concentration ratios of the two products (TCA plus TCE) to CH utilized were 1:1. In the NAD(P)(+)-linked reaction TCA was the predominant product and its amount was compatible with that of TCE plus NAD(P)H produced, whereas in the NAD(P)H-linked reaction equal amounts of TCA and TCE were formed and the cofactor was little oxidized. These results suggest that the enzyme oxidized the hydrated aldehydes to TCA with NAD(P)+ as the cofactor and reduced the unhydrated aldehyde to TCE with NAD(P)H. The steady-state kinetic measurements in the NADP(+)-linked CH oxidation were consistent with an ordered Bi Bi mechanism which is the same as that for the secondary alcohol oxidation by the enzyme. The dehydrogenase activity was inhibited competitively with respect to CH by a secondary alcohol substrate, propan-2-ol. The CH and propan-2-ol dehydrogenase activities were similarly inactivated by 2,4,6-trinitrobenzene-sulfonate, and NADP(H), several cofactor analogs and a cofactor-competitive inhibitor, Cibacron blue dye, protected against the inactivation, which suggest that lysine residues are essential for catalysis.