Human monoamine oxidase A gene determines levels of enzyme activity.

Monoamine oxidase (MAO) is a critical enzyme in the degradative deamination of biogenic amines throughout the body. Two biochemically distinct forms of the enzyme, A and B, are encoded in separate genes on the human X chromosome. In these studies we investigated the role of the structural gene for MAO-A in determining levels of activity in humans, as measured in cultured skin fibroblasts. The coding sequence of the mRNA for MAO-A was determined by first-strand cDNA synthesis, PCR amplification, and direct dideoxy sequencing. Two single-basepair substitutions were observed in cDNAs from cells with a 30-fold difference in activity levels. These two substitutions were in the third base of a triplet codon and hence did not affect the deduced amino acid sequence but did affect the presence or absence of restriction-enzyme sites for EcoRV and Fnu4HI, which could be elucidated on PCR fragments derived from genomic DNA or cDNAs. A third polymorphism for MspI in the noncoding region of the MAOA gene was also evaluated by Southern blot analysis using genomic DNA. Statistically significant associations were observed between the alleles for MAOA and levels of MAO activity in human male fibroblast lines. This association indicates that the MAOA gene itself is a major determinant of activity levels, apparently, in part, through noncoding, regulatory elements.     

Enhanced selectivity of pargyline as an inhibitor of type B monoamine oxidase in harmaline-treated rats

Pargyline, a slightly selective inhibitor of type B monoamine oxidase (MAO), inhibited phenylethylamine oxidation by 88 ± 1% and 81 ± 1% in rat brain and liver, respectively, at 24 hrs after injection of a 30 mg/kg i.p. dose. Serotonin oxidation was inhibited to a lesser extent, 68 ± 4% and 68 ± 2%, respectively, in brain and liver. In rats treated with harmaline, a short-lasting reversible MAO inhibitor selective for type A MAO, the inhibition of phenylethylamine oxidation after pargyline injection still occurred but the inhibition of serotonin oxidation was prevented. These results illustrate that a selective MAO inhibitor can be used to enhance the selectivity of an irreversible inhibitor, presumably by occupying active sites on a certain form of MAO temporarily and thereby preventing its inactivation. In heart, inhibition of both phenylethylamine and serotonin oxidation by pargyline was prevented by harmaline; this finding supports other evidence that phenylethylamine is metabolized by type A MAO in rat heart.     

An enzyme immunoassay for the measurement of human monoamine oxidase B protein.

A heterologous, competitive, solid phase ELISA has been developed which can measure monoamine oxidase (MAO) B concentration (both inactive and active) in human platelets and other tissue extracts. The assay is based on competition between a soluble form of MAO and MAO bound to a solid phase for binding to a limiting amount of a MAO B-specific monoclonal antibody, 3F12/G10. It utilises a crude and easily prepared sample of human liver mitochondrial membranes as the source of solid phase MAO. Optimal assay conditions allow detection of MAO B down to at least 0.5 ng (4.18 fmol; 6.25 ng/ml) of protein. As the assay is sensitive and simple to operate, it will allow a systematic assessment of the role of platelet MAO concentrations in the aetiology of psychiatric and neurologic conditions.     

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B

Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.     

Identification of novel monoamine oxidase B inhibitors by structure-based virtual screening

Parkinson’s disease is a severe debilitating neurodegenerative disorder. Recently, it was shown that the peroxisome proliferating-activator receptor-γ agonist pioglitazone protected mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity due to its ability to inhibit monoamine oxidase B (MAO-B). Docking studies were initiated to investigate pioglitazone’s interactions within the substrate cavity of MAO-B. Modeling studies indicated that the thiazolidinedione (TZD) moiety was a likely candidate for its specificity to MAO-B. To explore this potential novel MAO-B scaffold, we performed a structure-based virtual screen to identify additional MAO-B inhibitors. Our search identified eight novel compounds containing the TZD-moiety that allowed for a limited study to identify structural requirements for binding to MAO-B. Inhibition assays identified two TZDs (A6355 and L136662) which were found to inhibit recombinant human MAO-B with IC₅₀ values of 82 and 195 nM, respectively.     

Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods

Recently, evidence has been published which suggests that [Husain, M., Edmondson, D. E., & Singer, T.P. (1982) Biochemistry 21, 595-600] monoamine oxidase [amine:oxygen oxidoreductase (MAO), EC 1.4.3.4] deaminates phenylethylamine and benzylamine via two distinct kinetic pathways which involve either binary or ternary complex formation, respectively. These conclusions were drawn largely from stopped-flow kinetic analysis performed on purified enzyme removed from its native membrane and in the presence of the inhibitory detergent Triton X-100. In this study, d-amphetamine and alternative substrates were used as steady-state probes of the kinetics of deamination by the B form of human brain MAO using native membrane-bound enzyme. Initial velocity studies showed mixed-type patterns for amphetamine inhibition of phenylethylamine, tryptamine, and tyramine when either amine or oxygen was the varied substrate. Slope and intercept vs. amphetamine concentration replots were linear in all cases except for phenylethylamine (hyperbolic); Ki values obtained from linear replots of slope or intercept values were comparable. In contrast, amphetamine was a competitive inhibitor of benzylamine deamination when amine concentration was varied and uncompetitive when oxygen concentration was varied; slope and intercept replots were linear for both. When benzylamine was the alternative substrate inhibitor and tyramine and tryptamine deamination was measured, mixed-type inhibition patterns were obtained when either amine or oxygen concentration was varied; replots of slope and intercept were linear in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a monoamine oxidase B selective inhibitor from tobacco smoke

It is well established that tobacco smokers have reduced levels of monoamine oxidase activities both in the brain and peripheral organs. Furthermore, extensive evidence suggests that smokers are less prone to develop Parkinson's disease. These facts, plus the observation that inhibition of monoamine oxidase B protects against the parkinsonian inducing effects of the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, have prompted studies to identify monoamine oxidase inhibitors in the tobacco plant and tobacco cigarette smoke. Our previous efforts on cured tobacco leaf extracts have led to the characterization of 2,3,6-trimethyl-1,4-naphthoquinone, a non-selective monoamine oxidase inhibitor, and farnesylacetone, a selective monoamine oxidase B inhibitor. We now have extended these studies to tobacco smoke constituents. Fractionation of the smoke extracts has confirmed and extended the qualitative results of an earlier report [J. Korean Soc. Tob. Sci.1997, 19, 136] demonstrating the inhibitory activity of the terpene trans,trans-farnesol on rat brain MAO-B. In the present study, K(i) values for the inhibition of human, baboon, monkey, dog, rat, and mouse liver MAO-B have been determined. Noteworthy is the absence of inhibitory effects on human placental MAO-A and beef liver MAO-B. A limited structure-activity relationship study of analogs of trans,trans-farnesol is reported. Although the health hazards associated with the use of tobacco products preclude any therapeutic opportunities linked to smoking, these results suggest the possibility of identifying novel structures of compounds that could lead to the development of neuroprotective agents.     

2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase B inhibitors

2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular docking provided insight into the binding mode of these inhibitors and rationalized their different potencies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible α-methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or MAO-B. The best K_i values were in the 10^?8 M range, with selectivities towards human MAO-B exceeding 2000-fold.     

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine oxidase B

It has recently been reported that nitrile containing compounds frequently act as potent monoamine oxidase B (MAO-B) inhibitors. Modelling studies suggest that this high potency inhibition may rely, at least in part, on polar interactions between nitrile functional groups and polar moieties within the MAO-B substrate cavity. In an attempt to identify potent and selective inhibitors of MAO-B and to contribute to the known structure–activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC₅₀ values in the nanomolar range. In general, the sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding sulfanylbenzonitrile homologues. Among the compounds evaluated, 4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC₅₀ = 0.025 μM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease.     

Human monoamine oxidase enzyme inhibition by coffee and beta-carbolines norharman and harman isolated from coffee

Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recent epidemiological studies have consistently shown that coffee drinkers have an apparently lower incidence of Parkinson's disease (PD), suggesting that coffee might somehow act as a purported neuroprotectant. In this paper, "ready to drink" coffee brews exhibited inhibitory properties on recombinant human MAO A and B isozymes catalyzing the oxidative deamination of kynuramine, suggesting that coffee contains compounds acting as MAO inhibitors. MAO inhibition was reversible and competitive for MAO A and MAO B. Subsequently, the pyrido-indole (beta-carboline) alkaloids, norharman and harman, were identified and isolated from MAO-inhibiting coffee, and were good inhibitors on MAO A (harman and norharman) and MAO B (norharman) isozymes. beta-carbolines isolated from ready-to-drink coffee were competitive and reversible inhibitors and appeared up to 210 microg/L, confirming that coffee is the most important exogenous source of these alkaloids in addition to cigarette smoking. Inhibition of MAO enzymes by coffee and the presence of MAO inhibitors that are also neuroactive, such as beta-carbolines and eventually others, might play a role in the neuroactive actions including a purported neuroprotection associated with coffee consumption.     

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 g (wet weight) of cells (0.5 L of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molecular mass of 59.228 ± 0.064 kDa, which agrees with the calculated molecular weight of 59.172 kDa for the rMAOB protein sequence assuming one mole of covalent FAD per mole of the enzyme. Consistent with the MALDI-MS data, purified rMAOB shows a single band near 60 kDa in Coomassie-stained SDS–PAGE gel as well as on Western blot analyses performed using antisera raised against human MAOA and BSA-conjugated FAD. A partial amino acid sequence of the purified protein is confirmed to be that of the wild type rMAOB by in-gel trypsin digestion and MALDI-TOF-MS analyses of the liberated peptide fragments. Steady state kinetic data show that purified rMAOB exhibits a K_m(amine) of 176 ± 15 μM and a k_cat of 497 ± 83 min⁻¹ for benzylamine oxidation, and a K_m(O₂) of 170 ± 10 μM. Kinetic parameters obtained for purified rMAOB are compared with those reported earlier for recombinant human liver MAOB expressed in P. pastoris.     

Novel sulfanylphthalimide analogues as highly potent inhibitors of monoamine oxidase B

Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression and neurodegenerative diseases, respectively. Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5-sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC₅₀ values in the nanomolar range. The most potent inhibitor, 5-(benzylsulfanyl)phthalimide, exhibits an IC₅₀ value of 0.0045 μM for the inhibition of MAO-B with a 427-fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy.     

Substrate and inhibitor specificities for human monoamine oxidase A and B are influenced by a single amino acid

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for beta-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for beta-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also showed a distinct shift in sensitivity for the MAO A- and B-selective inhibitors Ro 41-1049 and Ro 16-6491. Mutant pair MAO A-T245I and MAO B-I236T and mutant pair MAO A-D328G and MAO B-G319D reduced catalytic activity but did not alter specificity. Our results indicate that Ile-335 in MAO A and Tyr-326 in MAO B play a critical role in determining substrate and inhibitor specificities in human MAO A and B.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

A novel and selective monoamine oxidase B substrate

Cyclic five- and six-membered tertiary allylamines constitute a unique class of monoamine oxidase substrates that undergo a net two-electron alpha-carbon oxidation to form the cyclic, conjugated eniminium metabolites. The corresponding saturated pyrrolidinyl and piperidinyl systems are not substrates for this flavoenzyme system. In an attempt to evaluate possible contributions that pi-orbital stabilization of the putative alpha-carbon radical intermediates may play in the catalytic pathway, we have examined the substrate properties of 3-methyl-6-phenyl-3-aza-bicyclo[4.1.0]heptane, the 3,4-cyclopropyl analog of the selective monoamine oxidase B substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results, which document the first reported example of a saturated, cyclic tertiary amine with monoamine oxidase substrate properties, are consistent with alpha-carbon radical stabilization as a contributing factor in the catalytic pathway.     

1,3,4-Oxadiazol-2-ylbenzenesulfonamides as privileged structures for the inhibition of monoamine oxidase B

The present study investigates the monoamine oxidase (MAO) inhibition properties of a series of ten 5-aryl-1,3,4-oxadiazol-2-ylbenzenesulfonamides. The target compounds were synthesized by dehydration of the corresponding N,N′-diacylhydrazines with phosphorus oxychloride to yield the 1,3,4-oxadiazole cycle with concomitant transformation of the sulfonamide to the sulfonyl chloride group. Treatment with aqueous ammonia in acetonitrile regenerated the target sulfonamides. The results of the enzymology document that these compounds are potent and specific MAO-B inhibitors with the most potent compound exhibiting an IC₅₀ value of 0.0027 µM. An analysis of the structure-activity relationships shows that the 4-benzenesulfonamides are significantly more potent MAO-B inhibitors than the corresponding 3-benzenesulfonamides, and that the corresponding N,N′-diacylhydrazine synthetic precursors are weak MAO inhibitors. Although MAO inhibition by oxadiazole compounds are known, this is the first report of nanomolar MAO inhibition potencies recorded for sulfonamide derivatives. MAO-B specific inhibitors such as those discovered here may be of interest in the treatment of neurodegenerative disorders such as Parkinson’s disease.     

Partial amino acid sequence analysis of human placenta monoamine oxidase A and bovine liver monoamine oxidase B

We have prepared peptide maps from human placenta monoamine oxidase type A (MAO-A) and bovine monoamine oxidase type B (MAO-B) and determined the amino acid sequences of 21 of these peptides. These sequences have been compared to the cDNA deduced amino acid sequences of human MAO-A and -B. A result of special interest is the identification of two sets of MAO-A peptides which have sequences different from those deduced from cDNA sequences. This observation is consistent with the notion that MAO-A may be composed of at two subunits which are similar but not identical in primary amino acid sequence.