The evaluation of isatin analogues as inhibitors of monoamine oxidase

The small molecule, isatin, is a well-known reversible inhibitor of the monoamine oxidase (MAO) enzymes with IC₅₀ values of 12.3 and 4.86 μM for MAO-A and MAO-B, respectively. While the interaction of isatin with MAO-B has been characterized, only a few studies have explored structure–activity relationships (SARs) of MAO inhibition by isatin analogues. The current study therefore evaluated a series of 14 isatin analogues as in vitro inhibitors of human MAO-A and MAO-B. The results indicated good potency MAO inhibition for some isatin analogues with five compounds exhibiting IC₅₀ < 1 μM. 4-Chloroisatin ( 1b ) and 5-bromoisatin ( 1f ) were the most potent inhibitors with IC₅₀ values of 0.812 and 0.125 μM for MAO-A and MAO-B, respectively. These compounds were also found to be competitive inhibitors of MAO-A and MAO-B with K_i values of 0.311 and 0.033 μM, respectively. Among the SARs, it was interesting to note that C5-substitution was particularly beneficial for MAO-B inhibition. MAO inhibitors are established drugs for the treatment of neuropsychiatric and neurodegenerative disorders, while potential new roles in prostate cancer and cardiovascular disease are being investigated.     

A monoclonal antibody elicited to human platelet monoamine oxidase. Isolation and specificity for human monoamine oxidase B but not A

We have isolated a mouse monoclonal antibody to human platelet monoamine oxidase (MAO) B. The antibody (MAO-1C2) was isolated from a fusion of mouse myeloma P3/X63 Ag8 to spleen cells from a BALB/c mouse immunized with a partially purified platelet preparation in which an estimated 21-31% of the protein was [3H]pargyline-labeled MAO B. The antibody indirectly immunoprecipitates both [3H]pargyline-labeled, catalytically inactive human MAO B, and unlabeled, catalytically active human MAO B. Binding of the antibody to MAO B has no detectable effect on catalytic activity. MAO-1C2 is specific for human MAO B, and fails to immunoprecipitate MAO A indirectly from human placenta or liver. Its ability to immunoprecipitate human MAO B but not MAO A from extracts of human liver provides a convenient technique for separating the two forms of the enzyme for comparative studies. The antibody does not recognize mouse liver MAO B, suggesting that the determinant is not universally expressed on MAO B from all species.     

Purification and some properties of porcine brain mitochondrial monoamine oxidase B.

Monoamine oxidase was isolated from pig brain mitochondria in a highly purified state by the following procedures: treatment with Ca-phosphate gel, column chromatography on DEAE-cellulose, mercurial-Sepharose, and hydroxyapatite chromatography and gel filtration on Bio-Gel A-1.5m. The properties of the enzyme disclosed that it is a monoamine oxidase of the B-type. The FAD present in the enzyme was covalently attached to the protein and it was shown to react with S-N,N-dimethylamino-1-propyne which is indicative of a reaction with the N-⁵ position of the isoalloxazine ring of FAD. The flavin content from spectral data and from the ¹⁴C-content of the enzyme inactivated with labeled 3-N,N-dimethylamino-1-propyne yielded a FAD content of 1 mole/104,000 g of protein. The subunit molecular weight from SDS-disc electrophoresis yielded a molecular weight of 52,000. Thus, it was concluded that the brain monoamine oxidase probably has two identical subunits, only one of which contained covalently bonded FAD.     

Localization of monoamine oxidase A and B and semicarbazide-sensitive amine oxidase in human peripheral tissues

Monoamine oxidase (MAO) A and B and semicarbazide-sensitive amine oxidase (SSAO) localizations in peripheral human tissues were compared by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3) and a rabbit polyclonal anti-bovine SSAO antibody. Immunoreactivities of the samples, obtained from 6 routine autopsy cases, showed different distributions in the tissues studied (heart, lung, duodenum, liver, pancreas, spleen, thyroid gland, adrenal gland and kidney). The relative MAO-A, MAO-B and SSAO distributions indicated a widespread distribution of these enzymes in the human body that is characterized by a matching cellular pattern in only few tissues. These differences suggest that each amine oxidase may play a specific function in, at least some, peripheral tissues.     

Development of benzylamine oxidase and monoamine oxidase A and B in man

We describe the widespread distribution of the enzymes benzylamine oxidase (BzAO) and monoamine oxidase A and B (MAO A, MAO B) in human tissues at three stages of development: fetal, neonatal and adult. Relatively low activity of each is present in fetal tissues, but the specific activities of BzAO and MAO B in fetal liver are similar to those in the adult, suggesting that these enzyme systems are functionally mature in the liver at 20 weeks gestation. Specific activity of BzAO in the lung, twice as high in the adult as in the fetus, reaches adult value at birth. In fetal brain, lung, aorta and digestive tract, MAO A emerges before MAO B. Estimation of total activity developed by tissue or organ sheds a new light on the importance of each enzyme in body economy and shows skeletal muscle to be by far the most active whole body source of MAO B, whilst the liver has the highest total and specific activity of MAO A. A similar approach clearly demonstrates that BzAO is essentially a tissue rather than a plasma enzyme, which tends to predominate in blood vessel walls.     

Inhibition of monoamine oxidase by analogues of amiloride

Amiloride analogues with nonaromatic substituents on the 5-amino group or different substituents on carbon-6 of the pyrazine ring were tested as inhibitors of monoamine oxidase A and B in rat brain homogenate. The inhibition was competitive and reversible. 5-(N,N-Tetramethylene)amiloride protected the A type in the homogenate against irreversible inhibition by clorgyline. A reciprocal relation was found to exist between inhibitory constants of 5-N-substituted amiloride analogues for monoamine oxidase A and the ratio of overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from the isolated rat tail artery incubated in the presence of a 50 microM concentration of the analogue, when the tissue was exposed to 10 microM tyramine. The 5-amino group appeared to be essential for inhibition of the A but not of the B type. Bell-shaped relations between inhibitory constants of 5-(N-alkyl)- and 5-(N,N-dialkyl)-substituted analogues and lengths of alkyl chains were different for each type. The presence of a methyl group in the alpha-position of the chain increased substantially the inhibitory constant for the A type. Halogen atoms as substituents on carbon-6 increased inhibitory constants for both types of the enzyme in the sequence: I less than Br less than Cl less than F. These findings are consistent with the existence of hydrophobic binding sites of restricted dimensions in both types of the enzyme.     

Determination of the absolute concentrations of monoamine oxidase A and B in human tissues

The concentrations of monoamine oxidase-A and -B were determined in homogenates of human cerebral cortex, caudatus and placenta and in human platelet-rich plasma and platelet membranes by determining the specific binding of tritium-labelled pargyline. The concentrations of the two enzyme forms were similar in both human brain regions examined. Determinations of the minimum quantities of clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition of enzyme activity was found to give overestimates of the amounts of monoamine oxidase-A and -B present due to nonspecific binding of these inhibitors.     

Styrene inhibits monoamine oxidase A, but not monoamine oxidase B in monkey brain mitochondria

The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.     

In vitro metabolism of citalopram by monoamine oxidase B in human blood.

The metabolism of the antidepressant citalopram (CIT) by monoamine oxidase B (MAO-B) was studied in vitro. In incubations with blood of nine healthy volunteers R-(P=0.015) and S-(P=0.0034) CIT propionic acid (CITPROP) production was correlated with the number of platelets. S-CITPROP production was 5.6 times higher than R-CITPROP production and in incubations containing the MAO-B inhibitor deprenyl, racemic CITPROP production was diminished to 9.1%. To our knowledge, this is the first time that MAO-B activity in blood is shown with an antidepressant as substrate. As MAO is strongly expressed in human brain, this observation suggests that this enzymatic system may be implicated in drug metabolism in the CNS.     

Modification of brain mitochondrial monoamine oxidase activities by hydroxyethylhydrazide of cyanoacetic acid and some other monoamine oxidase inhibitors.

Treatment of bovine brain mitochondrial membrane fragments with hydroxyethylhydrazide of cyanoacetic acid (HECA)—a hydrazine monoamine oxidase inhibitor in molecule of which an electronaccepting group is present—not only inhibited the monoamine oxidase activity but also induced appearance (or significant increase) of properties to catalyze deamination of various nitrogenous bases (cadaverine, histamine and others) which do not belong to the category of monoamine oxidase substrates. This phenomenon was prevented by pretreatment of the mitochondria with low concentrations of clorgyline—a selective inhibitor of monoamine oxidases of type A—but not by deprenil which is a selective inhibitor for the monoamine oxidases of type B. The data obtained suggest that HECA which inhibits the monoamine oxidase activity slowly (due to a time-dependent process) binds the active sites of monoamine oxidases of the type A and then induces qualitative modification (transformation) of their catalytic properties.     

Inactivation of purified human recombinant monoamine oxidases A and B by rasagiline and its analogues

The inactivation of purified human recombinant monoamine oxidases (MAO) A and B by rasagiline [N-propargyl-1(R)-aminoindan] and four of its analogues [N-propargyl-1(S)-aminoindan (S-PAI), 6-hydroxy-N-propargyl-1(R)-aminoindan (R-HPAI), N-methyl-N-propargyl-1(R)-aminoindan (R-MPAI), and 6-(N-methyl-N-ethyl carbamoyloxy)-N-propargyl-1(R)-aminoindan (R-CPAI)] has been investigated. All compounds tested, with the exception of R-CPAI, form stoichiometric N(5) flavocyanine adducts with the FAD moiety of either enzyme. No H(2)O(2) is produced during either MAO A or MAO B inactivation, which demonstrates that covalent addition occurs in a single turnover. Rasagiline has the highest specificity for MAO B, as demonstrated by a 100-fold higher inhibition potency (k(inact)/K(i)) compared to MAO A, with the remaining compounds exhibiting lower isozyme specificities. MAO B and MAO A are more selective for the R-enantiomer (rasagiline) compared to the S-enantiomer (S-PAI) by 2500-fold and 17-fold, respectively. Differences in UV/vis and CD spectral data of the complexes of the studied compounds with both MAO A and MAO B are interpreted in light of crystallographic data of complexes of MAO B with rasagiline and its analogues (Binda, C.; et al. J. Med. Chem. 2004, 47, 1767-1774.