Inactivation of monoamine oxidase A by the monoamine oxidase B inactivators 1-phenylcyclopropylamine, 1-benzylcyclopropylamine, and N-cyclopropyl-alpha-methylbenzylamine

Three known mechanism-based inactivators of beef liver mitochondrial monoamine oxidase (MAO) B are tested as inactivators of human placental mitochondrial MAO A. 1-Phenylcyclopropylamine (1-PCPA), 1-benzylcyclopropylamine (1-BCPA), and N-cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) are time-dependent irreversible inactivators of MAO A. The KI values for 1-PCPA and N-C alpha MBA, analogues of the MAO B substrate benzylamine, are much higher with MAO A than with MAO B. Evidence is presented to show that 1-PCPA inactivates MAO A by attachment to the flavin cofactor, unlike the reaction with MAO B in which 1-PCPA can attach to both a cysteine residue and the flavin [Silverman, R.B., & Zieske, P.A. (1985) Biochemistry 24, 2128-2138]. The reaction of 1-BCPA with MAO A was too slow to study in detail. N-C alpha MBA exhibits the same properties toward inactivation of MAO A that it does for inactivation of MAO B. Attachment in both cases is shown to be to one cysteine residue per enzyme molecule. The results with 1-PCPA indicate that the active site topographies of MAO A and MAO B are different. The ability of N-C alpha MBA to undergo attachment to a cysteine residue in both MAO A and MAO B may lead the way toward peptide mapping of the two isozymes in order to determine differences in their primary structures.     

Spectrometric evidence for the flavin-1-phenylcyclopropylamine inactivator adduct with monoamine oxidase N

1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with 1-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N(5)-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin-inactivator adduct. The incorporation of two (13)C labels into the inactivator, [2,3-(13)C(2)]-1-PCPA, followed by analysis using on-line liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin-inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N(5)-3-oxo-3-phenylpropyl-FAD.     

Facile synthesis of substituted trans-2-arylcyclopropylamine inhibitors of the human histone demethylase LSD1 and monoamine oxidases A and B

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.     

MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity

In eukaryotes, DNA is wrapped around proteins called histones and is condensed into chromatin. Post-translational modification of histones can result in changes in gene expression. One of the most well-studied histone modifications is the methylation of lysine 4 on histone H3 (H3K4). This residue can be mono-, di- or tri-methylated and these varying methylation states have been associated with different levels of gene expression. Understanding exactly what the purpose of these methylation states is, in terms of gene expression, has been a topic of much research in recent years. Enzymes that can add (methyltransferases) and remove (demethylases) these modifications are of particular interest. The first demethylase discovered, LSD1, is the most well-classified and has been implicated in contributing to human cancers and to DNA damage response pathways. Currently, there are limited methods for accurately studying the activity of demethylases in vitro or in vivo. In this work, we present MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation), a quantitative method for studying the activity of demethylases capable of removing mono- and di-methyl marks from lysine residues. We focus specifically on LSD1 due to its potential as a prime therapeutic target for human disease. This quantitative approach will enable better characterization of the activity of LSD1 and other chromatin modifying enzymes in vitro, in vivo or in response to inhibitors.Key words: LSD1, lysine demethylase, mass spectrometry, reductive methylation, monoamine oxidase (MAO) inhibitors     

Fluorinated phenylcyclopropylamines. Part 4: effects of aryl substituents and stereochemistry on the inhibition of monoamine oxidases by 1-aryl-2-fluoro-cyclopropylamines

A series of para-ring-substituted (E)- and (Z)-1-aryl-2-fluorocyclopropylamines were examined as inhibitors of recombinant human liver monoamine oxidase A (MAO A) and B (MAO B). Unlike the parent 1-phenylcyclopropylamine, which is a selective inhibitor of MAO B, both (E)- and (Z)-diastereomers of derivatives having fluorine at the 2-position of the cyclopropane ring were potent and selective irreversible inhibitors of MAO A. Both electron releasing groups (Me, OMe) and electron attracting groups (Cl, F) substituted in the para-position caused a modest increase in activity. Geminal difluoro-substitution caused a loss of potency of 100-fold compared to either (E)- or (Z)-monofluorinated analogue. Surprisingly, (1S,2R)-2-fluoro-1-phenylcyclopropylamine and the (1R,2S)-enantiomer were essential equally potent as inhibitors of MAO A and MAO B. None of the tested 1-aryl-2-fluorocyclopropylamines exhibited significant inhibition of tyramine oxidase.     

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol.2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k(cat)/K(m) values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-alpha-methylbenzylamine (N-CalphaMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CalphaMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.     

Mechanism of inactivation of monoamine oxidase by 1-phenylcyclopropylamine

1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since alpha,alpha-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when 1-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of beta-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of 1-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C]acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this beta-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of bovine plasma amine oxidase by haloallylamines

Various 2- and 3-haloallylamines were synthesized and evaluated as inhibitors of the quinone-dependent bovine plasma amine oxidase (BPAO). 3-Haloallylamines, which were previously found to be good inhibitors of the flavin-dependent mitochondrial monoamine oxidase (MAO), exhibited a time-dependent inactivation of BPAO, with the 2-phenyl analogs being more potent than the 2-methyl analogs. No plateau of enzyme activity loss was observed, suggestive of a lack of competitive partitioning to normal turnover. The (E)- and (Z)-2-phenyl-3-fluoro analogs were the most potent (low microM IC(50)s), with the corresponding 3-bromo and 3-chloro analogs being >10-fold less potent. In each case, the Z-isomers were more potent than the E-isomers, the reverse of the configurational inhibitory preference observed with MAO. In contrast to the 2-phenyl analogs, 3-phenyl-2(or 3)-chloroallylamines displayed a partitioning behavior, consistent with these being both substrates and inactivators of BPAO.     

Isolation of pure,catalytically active human liver monoamine oxidase B: Antibody complex

Monoamine oxidase B was purified from human liver mitochondria using a monoclonal antibody, MAO B-1C2, which recognizes monoamine oxidase B but not A. Triton X-100 extracts of mitochondria were incubated with purified MAO B-1C2 (IgG₁), and the catalytically active enzyme:antibody complex was isolated by affinity chromatography on Protein A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex revealed the presence of four polypeptide bands (monoamine oxidase B, 57,900 dalton; antibody heavy chain, 52,200 dalton; and two light chains, 29,400 and 27,700 dalton), and indicated a 1:1 stoichiometric ratio of enzyme to antibody. This method gave 154-fold purification of the enzyme from mitochondria.     

Effects of inhibitors on monoamine oxidase activity in perch (Perca flavescens) brain in vitro

1. Perch brain homogenates were incubated in vitro and monoamine oxidase (MAO) activity was determined fluorometrically, using a kynuramine substrate. 2. Clorgyline, harmaline and deprenyl inhibited MAO activity in a concentration-related manner, with single sigmoid inhibition curves, and the type A inhibitors harmaline and clorgyline were more effective than the type B inhibitor deprenyl. 3. Two types of inhibition were recognized in vitro; a fast-onsetting inhibition, similar to that produced by a reversible inhibitor, and a slow-onsetting inhibition, which is time- and concentration-dependent and presumably represents inactivation of the enzyme.     

Structure and catalytic mechanism of horseradish peroxidase. Regiospecific meso alkylation of the prosthetic heme group by alkylhydrazines

Horseradish peroxidase is inactivated in a time-, H2O2-, and concentration-dependent manner by phenylethyl-, ethyl-, and methylhydrazine. The pseudo- first order kinetic constants for these inactivation reactions at pH 7 are: phenylethyl (KI = 115 microM, kinact = 1.5 min-1, partition ratio = 11), ethyl (KI = 145 microM, kinact = 0.08 min-1, partition ratio = 32), and methyl (KI = 3000 microM, kinact = 0.12 min-1, partition ratio = 80). At pH 5, the constants for the phenylethyl reaction change to KI = 1540 microM and kinact = 0.86 min-1. A transient absorbance at approximately 830 nm, suggestive of an isoporphyrin intermediate, is seen during these reactions. The prosthetic heme is converted by each of the three alkylhydrazines into the corresponding delta-meso-alkylated heme. Complete inactivation of the enzymes by methyl-, ethyl-, and phenylethylhydrazine is associated with alkylation of 60-70, 70, and 90%, respectively, of the prosthetic heme groups. The absence of N-alkylation and the high specificity for the delta-meso position, even with agents as small as methylhydrazine, strengthen the proposal that electron abstraction is mediated by the heme edge rather than the ferryl oxygen of horseradish peroxidase.     

Evidence for a single catalytic binding site on human brain type B monoamine oxidase

The tricyclic antidepressant drug, amitriptyline, inhibited the B form of human brain mitochondrial monoamine oxidase (MAO) under normal atmospheric conditions in a noncompetitive manner when phenylethylamine (PEA) was used as substrate and competitively when benzylamine (BzNH2) was employed as substrate. In addition, it was also found that PEA and BzNH2 inhibited each other's degradation noncompetitively. Similar results have previously been reported with human platelet MAO. These data suggest that the catalytic binding sites for PEA and BzNH2 on the B form of human brain MAO may be different. Attempts were made to further distinguish these catalytic binding sites on the brain oxidase using the irreversible MAO inhibitors, pargyline and clorgyline. Though these drugs have considerably different affinities for the B form of the oxidase, the degree to which either compound inhibited PEA or BzNH2 deamination was essentially identical. When incubations were performed at elevated oxygen concentrations PEA and BzNH2 became mutually competitive inhibitors of each other's metabolism. Also at the higher levels of oxygen, amitriptyline inhibition of PEA deamination approached a competitive fashion. These results suggest that PEA and BzNH2 share a common catalytic binding site on the B form of MAO and, in addition, bind to an inhibitory site on the reduced form of the oxidase. Accordingly, the data indicate that amitriptyline also binds to both the oxidized and reduced forms of this human brain oxidase.     

Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER-) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.     

Fluorinated phenylcyclopropylamines. Part 3: Inhibition of monoamine oxidase A and B

Fluorinated phenylcyclopropylamines and alkylamines were examined as inhibitors of recombinant human liver monoamine oxidase A (MAO A) and B (MAO B). For a series of trans- and cis-2-fluoro-2-phenylcyclopropylamine analogues, the presence of fluorine attached to a cyclopropane ring was found to result in an increase in inhibitory activity towards both MAO A and B. In addition, p-substitution of electron-withdrawing groups such as Cl and F in the aromatic ring of the trans-isomers increased the inhibition of both enzymes. (1S,2S)-2-Fluoro-2-phenylcyclopropylamine was a more potent inhibitor of both MAO A and B than was the (1R,2R)-enantiomer, indicating that the presence of fluorine has no influence on the enantioselectivity of MAO inhibition, since a similar effect of stereochemistry has been reported for tranylcypromine. Interestingly, fluorination at the 2-position of 1-phenycyclopropylamine, which is known as a selective inhibitor of MAO B relative to MAO A, reversed the selectivity and resulted in a potent inhibitor selective for MAO A. All inhibitors showed time- and concentration-dependent inhibition for both enzymes, with the exception of trans-2-fluoro-2-phenylcyclopropyl ethylamine, which acts as a competitive and reversible MAO A selective inhibitor.