Inhibition of monoamine oxidase by C5-substituted phthalimide analogues

Literature reports that isatin as well as C5- and C6-substituted isatin analogues are reversible inhibitors of human monoamine oxidase (MAO) A and B. In general, C5- and C6-substitution of isatin leads to enhanced binding affinity to both MAO isozymes compared to isatin and in most instances result in selective binding to the MAO-B isoform. Crystallographic and modeling studies suggest that the isatin ring binds to the substrate cavities of MAO-A and -B and is stabilized by hydrogen bond interactions between the NH and the C2 carbonyl oxygen of the dioxoindolyl moiety and water molecules present in the substrate cavities of MAO-A and -B. Based on these observations and the close structural resemblances between isatin and its phthalimide isomer, a series of phthalimide analogues were synthesized and evaluated as MAO inhibitors. While phthalimide and N-aryl-substituted phthalimides were found to be weak MAO inhibitors, phthalimide homologues containing C5 substituents were potent reversible inhibitors of recombinant human MAO-B with IC(50) values ranging from 0.007 to 2.5 μM and moderately potent reversible inhibitors of recombinant human MAO-A with IC(50) values ranging from 0.22 to 9.0 μM. By employing molecular docking the importance of hydrogen bonding between the active sites of MAO-A and -B and the phthalimide inhibitors are highlighted.     

Characterization of multiple forms of carbonyl reductase from chicken liver.

Three enzyme forms (CR1, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17 beta-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbonyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.     

Cloning and bacterial expression of monomeric short-chain dehydrogenase/reductase (carbonyl reductase) from CHO-K1 cells.

Mammalian carbonyl reductase (EC 1.1.1.184) is an enzyme that can catalyze the reduction of many carbonyl compounds, using NAD(P)H. We isolated a cDNA of carbonyl reductase (CHO-CR) from CHO-K1 cells which was 1208 bp long, including a poly(A) tail, and contained an 831-bp ORF. The deduced amino-acid sequence of 277 residues contained a typical motif for NADP+-binding (TGxxxGxG) and an SDR active site motif (S-Y-K). CHO-CR closely resembles mammalian carbonyl reductases with 71-73% identity. CHO-CR cDNA had the highest similarity to human CBR3 with 86% identity. Using the pET-28a expression vector, recombinant CHO-CR (rCHO-CR) was expressed in Escherichia coli BL21 (DE3) cells and purified with a Ni2+-affinity resin to homogeneity with a 35% yield. rCHO-CR had broad substrate specificity towards xenobiotic carbonyl compounds. RT-PCR of Chinese hamster tissues suggest that CHO-CR is highly expressed in kidney, testis, brain, heart, liver, uterus and ovary. Southern blotting analysis indicated the complexity of the Chinese hamster carbonyl reductase gene.     

Immunological and enzymological localization of carbonyl reductase in ovary and liver of various species

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.     

靛红生物活性研究进展

靛红是一种重要的天然产物,广泛分布于动植物和人体内,具有多种生物活性,在生物体内起着重要的作用.本文对靛红在动物和人体内作用于神经系统、单胺氧化酶、利钠肽以及其抗肿瘤、抗衰老等方面的活性的研究进展进行了综述.     

Speciesabhängigkeit der Monoaminoxydase-Aktivität in verschiedenem Lebensalter

Summary The monoamine oxidase activity in ten species (man, dog, cat, rabbit, guinea pig, rat, hamster, mouse, chicken, goose) was histochemically studied in the myocardium, liver, kidney and psoas muscle in newborn and older individuals.An age-dependent increase of monoamine oxidase activity is established in the liver and kidney of man, dog, cat, guinea pig and hamster. In the psoas muscle of the rat the monoamine oxidase activity is consistently weak. In the myocardium only the rat shows an increase with age.     

Substrate specificity of three prostaglandin dehydrogenases

Studies on the substrate specificity, kcat/Km, and effect of inhibitors on the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase (9-ketoprostaglandin reductase) indicate that it is very similar to a human brain carbonyl reductase which also possesses 9-ketoprostaglandin reductase activity. These observations led to a comparison of three apparently homogeneous 15-hydroxyprostaglandin dehydrogenases with varying amounts of 9-ketoprostaglandin reductase activity: an NAD- and an NADP-linked enzyme from human placenta and an NADP-linked enzyme from rabbit kidney. All three enzymes are carbonyl reductases for certain non-prostaglandin compounds. The placental NAD-linked enzyme, which has no 9-ketoprostaglandin reductase activity, is the most specific of the three. Although it has carbonyl reductase activity, a comparison of the Km and kcat/Km for prostaglandin and non-prostaglandin substrates of this enzyme suggests that its most likely function is as a 15-hydroxyprostaglandin dehydrogenase. The results of similar comparisons imply that the other two enzymes may function as less specific carbonyl reductases.     

The effect of isatin (tribulin) on metabolism of indoles in the rat brain and pineal: In vitro and in vivo studies

Isatin (Tribulin) produced a dose-dependent inhibition of both MAO A and MAO B in broken cell preparations from rat brain and pineal. However, isatin administered in vivo (80–160 mg/kg) to the intact animal significantly increased brain, but not pineal, serotonin and did not affect 5HIAA or other indoles in either brain or pineal. Further, in vivo administration did not produce detectable MAO inhibition in either tissue. In pineal organ culture, addition of isatin up to 1mM had no influence on the concentrations of pineal indoles or the activities of monoamine oxidase or serotonin N-acetyltransferase. However, the diazepam augmentation of beta adrenergic induction of serotonin N-acetyltransferase activity was blocked by isatin. The results of these studies call into question the proposed role of isatin as an endogenous monoamine oxidase inhibitor but support a possible role as a benzodiazepine receptor blocker.     

Subcellular distribution of OM cytochrome b-mediated NADH-semidehydroascorbate reductase activity in rat liver

Tissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.     

Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats

trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent.     

Formation of the superoxide anion radical during the oxidation of biogenic amines catalyzed by mitochondrial monoamine oxidase

The studies on the activity of monoamine oxidase from human placenta, using 2-phenylethylamine as a substrate, corroborate the hypothesis on the possible superoxide radical generation upon FAD oxidation at the second (aerobic) stage of monoamine oxidase reaction. It has been shown that hydrogen peroxide, but not other activated O2 forms, was the end product of this reaction. No superoxide radical generation took place in such systems. And therefore, the induction of lipid peroxidation in the presence of catalase was impossible in mitochondrial membranes containing monoamine oxidase and amines oxidized by it.     

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.     

Isatin, an endogenous monoamine oxidase inhibitor, triggers a dose- and time-dependent switch from apoptosis to necrosis in human neuroblastoma cells

Isatin is an endogenous indole that is increased in stress, inhibits monoamine oxidase (MAO) B and improves bradykinesia and striatal dopamine levels in rat models of Parkinson's disease. Consequently, it has been suggested that isatin might be a possible treatment for Parkinson's disease although little is known about its effects on neural cell growth and survival. The aim of this study was to investigate the survival of dopaminergic human neuroblastoma (SH-SY5Y) cells following treatment with increasing concentrations of isatin. SH-SY5Y cells were exposed to isatin for defined time points, after which cell survival was determined by MTT assay. A combination of Annexin V binding and propidium iodide (PI) exclusion was used to distinguish apoptosis from necrosis in flow cytometry experiments and FACS profiles of permeabilised PI-labelled cells were employed for the assessment of cell cycle distribution. Isatin treatment (1-400 microM) for 24h induced a significant dose-dependent increase in MTT metabolism by SH-SY5Y cells in culture, but this was not due to an increase in cell division. At the higher concentrations (200-400 microm) isatin triggered cell death, although MTT metabolism was still increased in the culture, suggesting that surviving cells were hypermetabolic. Following a longer (48 h) exposure, isatin was found to cause cell death in a dose-dependent manner; at lower concentrations (50 microM), the predominant mode of cell death was apoptosis while at the highest concentration (400 microm) increasing numbers of necrotic cells were also evident. Thus, in dopaminergic SH-SY5Y cells isatin induces cell death in dose- and time-dependent manner. This death occurred as a continuum of survival, apoptosis and necrosis. Our results re-emphasise that caution should be exercised when considering high doses of isatin as a putative anti-Parkinson's disease therapeutic.     

Inhibition of monoamine oxidase by 3,4-dihydroxyphenyl L-alanine and its analogues

L-3,4-Dihydroxyphenylalanine (DOPA) was found to inhibit type A monoamine oxidase in human placental mitochondria. The inhibition proved to be noncompetitive with the substrate, kynuramine, and the inhibition was completely reversible. D-DOPA was found to inhibit monoamine oxidase in the same way, and the apparent Ki values of L- and D-DOPA were obtained to be 154 microM and 133 microM, respectively. L-alpha-Methyl-DOPA was found to inhibit the MAO activity competitively with the substrate, but studies with other analogues of DOPA revealed that the inhibition required an amino and a carboxyl group at alpha-position. The substitution of a hydroxy group at 3 or 4 position of catechol ring with a methoxy group was found to abolish the inhibition of the MAO activity. In addition to type A MAO in human liver and placental mitochondria, type B MAO in liver mitochondria was inhibited by L-DOPA, but type B MAO was less sensitive to L-DOPA. These results were discussed in terms of its possible regulation of the level of biogenic amines in the brain.     

Enzymatic oxidation of all-trans retinal to retinoic acid in rat tissues

The 100,000 x g supernatant (cytosolic) fraction of rat tissue homogenates catalyzes the oxidation of all-trans retinal to retinoic acid. Kidney, testis, and lung were the most active of the tissues examined. The presence of enzyme activity in liver and intestine could be detected only when a substrate concentration beyond the saturation point for retinal reductase was used. Spleen, brain, and plasma had no activity. Boiled supernatants did not catalyze the reaction. The enzymatic product was chemically and physically identified as retinoic acid. The cytosol of kidney tissue also catalyzed the conversion of retinol to retinoic acid. These data indicate that kidney tissue has the highest retinal oxidase activity and suggest that it may play a major role in the oxidative metabolism of retinol in the body.     

N-(1-Methyl)cyclopropylbenzylamine: A novel inactivator of mitochondrial monoamine oxidase

N-(1-Methyl)cyclopropylbenzylamine was synthesized and shown to inactivate pig liver mitochondrial monoamine oxidase. Inactivation is time-dependent, concentration dependent, protected by the substrate and product of the enzyme, and is not reversed by exhaustive dialysis. Unlike N-cyclopropylbenzylamine, the adduct formed between N-(1-methyl)cyclopropylbenzylamine and monoamine oxidase is stable to treatment with benzylamine. A mechanism for the inactivation is proposed.     

OCCURRENCE AND PROPERTIES OF MONOAMINE OXIDASE IN ADRENERGIC NEURONS

—Monoamine oxidase activity of peripheral organs of various species has been examined after surgical, chemical and immunological sympathectomy to assess the proportion of enzyme activity in adrenergic neurons and in extraneuronal cells. Significant falls in monoamine oxidase activity of vas deferens, submaxillary gland, iris and spleen were seen after sympathetic denervation although not in heart, small intestine and kidney. It was suggested that a correlation exists between the extent of the fall in monoamine oxidase activity after sympathectomy and the density of sympathetic innervation of the control organ. Studies of monoamine oxidase activity in vas deferens after inhibition with clorgyline suggested multiple forms of monoamine oxidase. Differences in inhibitor sensitivity, substrate specificity and thermal inactivation of monoamine oxidase in normal and denervated vas deferens were found and it was suggested that differences exist in the properties of the neuronal and extraneuronal monoamine oxidase.     

Histochemical evidence of monoamine oxidase activity in rats of different ages

Summary The histochemically detectable monoamine oxidase activity in certain organs of young and old rats is compared. Regardless of age, the activity is strong in the liver, faint in the skeletal muscle, and absent in the kidney. In the myocardium, however, the quantity of monoamine oxidase increases strongly with age. Its activity is manifest in the form of granular and diffuse formazan precipitates; both disappear after a preliminary treatment of the animals with a monoamine oxidase inhibitor. This finding indicates that the diffuse as well as the previously identified granular precipitates represent monoamine oxidase.     

Histochemical study of monoamine oxidase latency in the liver of the rat

Summary In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

Histochemical study of monoamine oxidase latency in the liver of the rat.

In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.