Inhibition of aldehyde reductase isoenzymes in human and rat brain.

Anticonvulsant drugs, such as barbiturates, open-chained analogues of barbiturates, glutethimide, succinimides and hydantoins have been tested in vitro as inhibitors of the isoenzymes of aldehyde reductase from human and rat brain. One major isoenzyme of both species is highly sensitive to the ionizable forms of these drugs containing the CONHCO grouping and a minimal lipophilic substitution. Differences between the isoenzymes were observed with respect to the absolute configuration of various succinimides as inhibitors. One isoenzyme of both species exerts activity with NADH as well as NADPH. The NADH-dependent activity of the human enzyme is inhibited in a noncompetitive way by NADP with a K_i-intercept of 2.2 × 10^?7 M. A mixed inhibition is obtained with the biogenic acid, 4-hydroxyphenylacetic acid, as inhibitor of the main human isoenzyme. The inhibition is uncompetitive up to [I] = 1 × 10^?3 M and yields a K_i value of 4.2 × 10^?4M.     

Inhibition of bovine brain aldehyde reductase by anticonvulsant compounds in vitro

The catalytic activity of partially purified NADPH-linked aldehyde reductase (alcohol: NADP oxidoreductase, EC 1.1.1.2) from bovine brain was markedly inhibited in vitro by anticonvulsant compounds. In general, the ability of these drugs to inhibit aldehyde reductase in vitro paralleled their anticonvulsant activity. Inhibition by various barbiturates, hydantoins or succinimides was non-competitive with either NADPH or aldehyde as the variable substrate, whereas the 2,4-oxazolidinediones produced a mixed type of inhibition. Inhibition by all ionizable compounds was found to vary with the pH of the reaction mixture, while the non-ionizable substances, paradione, trimethadione, methsuximide, 3-methyl-5-ethyl-5-phenylhydantoin, were not inhibitory. At pH 7.0 the inhibitor constants (K_i values) for phenobarbital, 5,5-diphenylhydantoin, 5,5-dimethyloxazolidinedione and ethosuximide were 1.2 × 10^?4 M, 1.7 × 10^?4 M, 4.7 × 10^?4 M and 5.5 × 10^?3 M respectively. The possibility that inhibition of brain NADPH-linked aldehyde reductase by these agents is concerned with their anticonvulsant actions is discussed.     

Inhibition of aldehyde reductase by aldose reductase inhibitors

A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been examined for their ability to inhibit rat kidney aldehyde reductase (EC 1.1.1.19, EC 1.1.1.20) versus rat lens aldose reductase (EC 1.1.1.21). All aldose reductase inhibitors examined inhibited aldehyde reductase to some extent both in the reductive reaction as determined with glyceraldehyde as substrate and NADPH as coenzyme, and in the oxidative reaction where L-gulonic acid was oxidized to D-glucuronic acid in the presence of NADP+. Of the inhibitors examined, 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dion e (Al1576) was the most potent inhibitor requiring only concentrations in the 10(-8) M range to inhibit 50% of the in vitro activity of rat kidney aldehyde reductase (IC50 value), whereas 3-dioxo-1-H-benz[de]isoquinoline-2(3H)-acetic acid (alrestatin) was the least potent inhibitor requiring concentrations in the 10(-5) M range. Both the reductive and oxidative steps appeared equally inhibited by these aldose reductases inhibitors. Moreover, all compounds appeared to inhibit either crude or highly purified rat kidney aldehyde reductase to essentially the same extent. Marked differences in the selectivity of these inhibitors, expressed as the ratio of IC50 values for rat kidney aldehyde reductase versus rat lens aldose reductase with glyceraldehyde as substrate, were observed with selectivity for aldose reductase ranging from ca. 2-fold for Al1576 to 119-fold for 3-(4-bromo-2-fluorobenzyl-4-oxo-3-phthalazine-1-ylacetic acid (Ponalrestat). Kinetic and competition studies suggest that these inhibitors interact with aldehyde reductase at a common site that is not identical to either the substrate or nucleotide binding site. These results suggest that the inhibitor binding sites of rat kidney aldehyde reductase and aldose reductase contain several common characteristics.     

Inhibitory effects of fidarestat on aldose reductase and aldehyde reductase activity evaluated by a new method using HPLC with post-column spectrophotometric detection

A new method to assay the activity of aldose reductase (AR) and aldehyde reductase (AHR) by high-performance liquid chromatography is described. The separation of AR and AHR from tissue extracts using an anion-exchange column was followed by chromatographic measurement of the activity in the elute. AR and AHR activity were expressed as the area under the peak obtained by post-column spectrophotometric detection of the decrease of coenzyme (NADPH) in each enzyme reaction. In the enzyme preparation from rat or human tissues obtained by this method, two active peaks were identified as AR and AHR. The correlation coefficient between the injection volume of the enzyme preparation from each tissue and each peak area was 0.998 or greater. In addition, the within-day preservation rate of AR or AHR activity from each tissue was over 95%. In a comparative study of fidarestat with other AR inhibitors using this method, it was confirmed that the inhibitory effect of fidarestat on AR activity from each rat tissue was more potent than that produced by sorbinil and equipotent to that of epalrestat and zenarestat. Fidarestat was also found to inhibit AR activity more potently than AHR activity in human erythrocytes. Therefore, this method is applicable to studies of the selective inhibition of AR or AHR by test compounds.     

四种药物对人脑醛糖还原酶的抑制作用

从人脑提纯了醛糖还原酶,应用酶动力学的方法研究了四种药物对此酶的抑制作用。结果表明索比尼尔,阿司他丁和苯巴比妥是人脑醛糖还原酶的完全性非竞争性抑制剂,四甲烯戊二酸是此酶的部分性非竞争性抑制剂。从抑制曲线及四种药物的抑制常数(Ki)可知,索比尼尔是人脑醛糖还原酶最有效的抑制剂,阿司他丁次之。根据苯巴比妥的Ki和它的毒性作用推测,此药似不宜以醛糖作为原酶的抑制剂应用于临床。     

Effects of benzodiazepines and valproic acid on brain aldehyde reductase and a proposed mechanism of anticonvulsant action

Benzodiazepines (clonazepam, diazepam, flurazepam, fosazepam, lorazepam, nitrazepam, oxazepam and R07-5205) were shown to inhibit the activity of brain aldehyde reductase obtained from DBA/2J mice with the ic₅₀ values (concentration of inhibitor at 50 per cent of control activity) ranging from 0.24 to 7.0 mM. ed₅₀ Values of these benzodiazepines for protection against maximal electroshock-induced convulsions were determined for DBA/2J mice which were pretreated with either saline or β-diethylaminoethyl diphenylpropylacetate (SKF-525A), an inhibitor of microsomal drug-metabolizing systems. Spearman rank order and Pearson correlation coefficients between the ic₅₀ values for inhibition of aldehyde reductase activity and the ed₅₀ values for protection against maximal electroshock-induced convulsions were calculated to be 0.62 and 0.82, respectively, for a group of eight benzodiazepines. When the animals were pretreated with SKF-525A, the correlation coefficients were 0.83 and 0.71, respectively. R_m values, indicators of relative lipid solubility, were measured for these benzodiazepines. Correlations between R_m values and ic₅₀ values or ed₅₀ values were not significant at the 95 per cent confidence level.Valproic acid inhibited DBA/2J mouse brain aldehyde reductase activity with an ic₅₀ value of 7 × 10^?5 M. Data presented in this study are consistent with the hypothesis that highly reactive aldehyde intermediates of biogenic amine metabolism may be implicated in anticonvulsant drug action.     

Purification and properties of aldose reductase and aldehyde reductase from EHS tumor cells

Engelbreth-Holm-Swarm (EHS) tumor cells were utilized as a model for investigating the production of basement membrane components. These cells contain two immunologically distinct NADPH-dependent reductases, aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2), which were purified to apparent homogeneity by a combination of procedures which included ammonium sulfate fractionation, Sephadex G-75 gel filtration, Matrex Gel Orange A affinity chromatography, and chromatofocusing on Pharmacia Mono P. The molecular weights of aldose and aldehyde reductases were estimated to be 38K and 40K, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed that both enzymes were capable of reducing a variety of aldehydes to their respective alcohols; however, only aldehyde reductase oxidized L-gulonic acid. Surprisingly, both enzymes showed similar reactivities with D-glucose and D-galactose, suggesting that both aldose and aldehyde reductases may contribute to sorbitol production in the EHS tumor cell. The activities of both enzymes were increased by the presence of sulfate ion, but chloride ion decreased the activity of aldose reductase. Both aldose and aldehyde reductases were inhibited by a series of structurally diverse aldose reductase inhibitors.     

Compensatory increase in synaptosomal aldehyde reductase activity in rat brain after chronic barbital treatment

The changes in brain aldehyde reductase (AIR) activities during long-term treatment of rats with barbital were studied. NADH-linked AIR activity in the synaptosomal fraction increased rapidly, and supernatant AIR activity rose later during barbital treatment. With respect to NADPH-linked AIR, two distinct K_m values were observed for p-nitrobenzaldehyde. The low-K_m enzyme had a higher K_i value than the high-K_m enzyme. Although the elevation of NADPH-linked AIR activity under the routine assay conditions was less remarkable, V_max values of the low-K_m enzyme were greatly increased in both the synaptosomal and the supernatant fractions by chronic barbital treatment. In addition, the K_i value of low-K_m AIR in synaptosomes was greater in barbital-treated animals than in control animals. K_m values were unchanged in either fraction by chronic barbital treatment. These data suggest that chronic barbital treatment resulted in a compensatory increase in the activity of AIR with low K_m, which is less sensitive to barbital and utilizes either NADH or NADPH, in synaptosomes.     

Nicotinamide adenine dinucleotide phosphate-decanaldehyde adduct as an inhibitor of beef brain NADP-linked aldehyde reductase.

The synthesis and characterization of the addition product of NADP and decanaldehyde are described. This adduct is an inhibitor of beef brain NADP-linked aldehyde reductase (Ki = 2.3 x 10(-6) M) along with the NADP adducts of butyraldehyde, phenylpropionaldehyde, and phenylacetaldehyde.     

"Biogenic" aldehyde metabolism relation to pentose shunt activity in brain

Biogenic amines, added to brain homogenates, were demonstrated to stimulate oxidative decarboxylation of glucose isotopically labeled at C-1. This effect was ascribed to the stimulation of the pentose phosphate shunt in brain and was found to depend on the monoamine oxidase (MAO)-catalyzed production of the aldehyde derivatives (biogenic aldehydes) of the biogenic amines. The stimulation produced by the amines and the aldehydes was shown to be inhibited by barbiturates, and the enzymes responsible for the stimulated metabolism of glucose were found to be present in the cytosol. Evidence is presented indicating that the stimulation produced by biogenic aldehydes depends on the oxidation of NADPH to NADP by aldehyde reductase present in brain cytosol. Acid derivatives of the biogenic amines [i.e. 5-hydroxyindoleacetic acid (5-HIAA)]were found to inhibit aldehyde-stimulated metabolism of glucose by the pentose phosphate shunt.     

Inhibition of ox brain glutamate dehydrogenase by perphenazine

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the antipsychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However, the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.     

Daunorubicin metabolism: estimation of daunorubicin reductase

1. Daunorubicin reductase, an enzyme ubiquitous in all mammalian tissues tested, converts daunorubicin, a cancer chemotherapeutic antibiotic, to daunorubicinol. A method for the estimation of daunorubicinol and daunorubicin reductase is described.2. Daunorubicin and daunorubicinol were quantitatively extracted from enzyme assay mixtures with 0.3 N HCl in 50% ethanol and then completely hydrolysed to their respective aglycones. The aglycones were extracted, concentrated into toluene, separated by silicic acid microcolumn chromatography, and estimated by spectrofluorimetry.3. Compared to previous methods, this method is highly reproducible and sensitive.     

人参皂苷对醛糖还原酶的抑制作用

目的糖尿病并发症与糖代谢的多元醇通路有关,而醛糖还原酶(aldose reductase,AR)是该通路的关键限速酶。本研究拟分析不同类型人参皂苷对AR的抑制作用,探讨其在糖尿病并发症治疗中的潜在应用价值。方法采用分光光度法测定不同类型人参皂苷对AR的抑制作用,并依据动力学曲线计算其半数抑制浓度((IC50));通过双倒数作图法确定人参皂苷对AR抑制作用的类型。结果人参皂苷Rb1、Rd、Rg1和Rg2对AR具有抑制作用,而人参皂苷Rb2、Rb3、Rc和Re的抑制作用不明显。进一步分析表明,人参皂苷Rd、Rg1和Rg2对AR的抑制作用强于"依帕司他",而Rb1的抑制作用较弱;另外,人参皂苷的浓度与其对AR的抑制作用正相关,且抑制类型均为反竞争性抑制。结论人参皂苷Rg2、Rg1、Rd对该AR具有较强的抑制作用,在糖尿病并发症治疗方面具有潜在的应用价值。     

Structure of aldehyde reductase holoenzyme in complex with the potent aldose reductase inhibitor fidarestat: implications for inhibitor binding and selectivity

Structure determination of porcine aldehyde reductase holoenzyme in complex with the potent aldose reductase inhibitor fidarestat was carried out to explain the difference in the potency of the inhibitor for aldose and aldehyde reductases. The hydrogen bonds between the active-site residues Tyr50, His113, and Trp114 and fidarestat are conserved in the two enzymes. In aldose reductase, Leu300 forms a hydrogen bond through its main-chain nitrogen atom with the exocyclic amide group of the inhibitor, which when replaced with a Pro in aldehyde reductase, cannot form a hydrogen bond, thus causing a loss in binding energy. Furthermore, in aldehyde reductase, the side chain of Trp220 occupies a disordered split conformation that is not observed in aldose reductase. Molecular modeling and inhibitory activity measurements suggest that the difference in the interaction between the side chain of Trp220 and fidarestat may contribute to the difference in the binding of the inhibitor to the enzymes.     

Effects of aldehyde dehydrogenase inhibitors on enzymes involved in the metabolism of biogenic aldehydes in rat liver and brain

The effects of the aldehyde dehydrogenase inhibitors disulfiram, coprine and cyanamide on enzymes involved in the metabolism of biogenic aldehydes in rat liver and brain were studied. Both liver and brain aldehyde dehydrogenase activities were significantly decreased in rats pretreated with these drugs. In the liver, the low-K_m aldehyde dehydrogenase activity was markedly decreased by all three drugs after 2 and 24 hr whereas only cyanamide inhibited the high-K_m enzymes. The brain ALDH-activity with a low acetaldehyde concentration was significantly decreased by coprine and cyanamide at both times tested, whereas disulfiram caused no change after 2 hr but an inhibition of 38% after 24 hr. The brain ALDH-activity with a high acetaldehyde concentration was significantly decreased by coprine and cyanamide but not by disulfiram. The activity of the substrate specific enzyme succinate semialdehyde dehydrogenase in brain was slightly but significantly decreased in rats pretreated with cyanamide but not in rats pretreated with disulfiram or coprine. None of the drugs caused any changes in the activities of aldehyde reductase and monoamine oxidase in brains in vivo. The activity of monoamine oxidase in liver was significantly decreased by coprine after 24 hr. In contrast to the effects obtained in vivo, disulfiram was found to be an inhibitor in vitro of brain succinate semialdehyde dehydrogenase and liver monoamine oxidase. Aldehyde reductase was slightly inhibited by both disulfiram and 1-aminocyclopropanol in vitro.     

The role of aldehyde oxidase in drug metabolism

INTRODUCTION: Aldehyde oxidases (AOXs) are molybdo-flavoenzymes with complex evolutionary profiles, as the number and types of active AOX genes vary according to the animal species considered. Humans and higher primates have a single functional AOX1 gene, while rodents are endowed with four AOXs. Along with the endoplasmic cytochrome P450 system (CYP450), cytoplasmic AOX1 is the major enzyme involved in the hepatic phase I metabolism of numerous xenobiotics. AREAS COVERED: The authors review literature to highlight the fact that aldehydes are not the only AOX substrates, as aza- and oxo-heterocycles, that represent the scaffold of many drugs, are also oxidized efficiently by these enzymes. Additionally, the ndefine the different complements of AOX isoenzymes expressed in humans and animal models used in drug metabolism studies and discuss the implications. Furthermore, the authors report on human AOX1 allelic variants that alter the activity of this enzyme. Finally, they discuss the factors of potential importance in controlling the functional activity of AOX1. EXPERT OPINION: There is evidence for an increasing relevance of AOX1 in the metabolism and clearance of new drugs, as measures aiming at controlling CYP450-dependent metabolism of prospective therapeutic agents are becoming routine. This calls for investigations into the biology, catalytic properties and substrate specificity of human AOX1.