the brain. MAO B inhibitors can also be used for the b-Carbolines are endogenous inhibitors of mono-prevention and adjunct treatment of Parkinson's disamine oxidase (MAO). The interaction of nine b-carbo-ease (1). In the last decade, extensive studies have led line derivatives and four 3,4-dihydro forms with purito the discovery of a new class of potent and selective fied MAO A was investigated. All the compounds tested reversible MAO inhibitors (see reviews, (2, 3)), includwere reversible competitive inhibitors selective for ing moclobemide, brofaromine, Ro 41-1049, and toloxa-MAO A, in agreement with previous studies on memtone for MAO A and Ro 16-6491, Ro 19-6327, and oxadibrane preparations. The oxidation of kynuramine by azolone derivatives (4, 5) for MAO B. Both forms of MAO A in the presence of the more effective inhibitors the enzyme oxidize primary, secondary, and tertiary showed a lag period before reaching the steady state. amines, but they are distinguished by differences in In general, the 1-methyl and 7-methoxy substituents their substrate and inhibitor specificities (for a review, increased the potency. Harmine, 2-methylharminium, see ). They are separate enzymes encoded by differ-2,9-dimethylharminium, and harmaline were the most ent genes and characterized by different biochemieffective inhibitors of the purified MAO A, with low K i cal and immunological properties (8, 9). MAO A and values of 5, 69, 15, and 48 nM, respectively. The inhibi-MAO B are expressed in different proportions in differtors interacted with the covalently bound flavin to inent cell types and their expression varies in developduce distinct spectral changes, the magnitude of ment and ageing. These differences imply specific physwhich correlated with the efficacy of the inhibition. iological roles for each, hence the significance of specific The more effective inhibitors could be in situ inhibiinhibition. tors of MAO A. ᭧ 1997 Academic Press, Inc. b-Carbolines (BCs) are heterocyclic, dehydrogenated Key Words: monoamine oxidase; b-carbolines; monoderivatives of tryptophan. When methylated on the niamine oxidase inhibitors; difference spectrum. trogen in the 2-position, the derivatives are structural analogs of the N-methyl-4-phenylpyridinium ion (MPP / ). Certain N-methylated derivatives display mi-Monoamine oxidases (MAO, EC 1.4.3.4) 3 A and B tochondrial inhibitory properties and neurotoxic effects catalyze the degradation of neuroactive and vasoactive (10, 11), similar to the effect of MPP / on the mitochonamines in the central nervous system and in peripheral drial respiratory chain (12, 13). In addition to the eftissues. Inhibition of MAO by antidepressant drugs refects on mitochondria, MPP / and its analogs are also sults in the build-up of amines such as serotonin in reversible, competitive, product inhibitors of MAO A (14). The BCs, such as [ 3 H]harmaline (15) and more recently [ 3 H]harman (16) have been used as specific has been used for quantitative autoradiography in rat 2 To whom correspondence and reprint requests should be addressed at School of Biological and Medical Sciences, University of brain (17). The MAO inhibitory properties of the tet-St. Andrews, Irving Building, North Street, St. Andrews KY16 9AL, rahydro-b-carbolines and of a few of the aromatic bhave been studied in beef liver membrane 3 Abbreviations used: MAO, monoamine oxidase; BC, b-carboline; preparations, which contain mainly MAO B (18). That MPP / , 1-methyl-4-phenylpyridinium ion; DTE, dithioerythritol; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.

The structure of monoamine oxidase B [Nat. Struct. Biol. 9 (2002) 22] revealed three aromatic amino acid residues within contact distance of the flavin cofactor and a large number of aromatic residues in the substrate binding site. Circular dichroism (CD) spectroscopy can detect alterations in the environment of aromatic residues as a result of ligand binding or redox changes. CD spectra of MAO A indicate that a small inhibitor such D-amphetamine perturbs the aromatic residues very little, but binding of the larger pirlindole (2,3,3a,4,5,6-hexahydro-8methyl-1H-pyrazino[3,2,1-j,k]carbazole hydrochloride) causes spectral changes consistent with the alteration of the environment of tyrosine and tryptophan residues in particular. Reduction of the flavin cofactor induces large enhancement of the CD signals in the aromatic region (260 -310 nm). When covalent modification of the flavin by clorgyline accompanies reduction, the perturbation is even greater. In contrast to the static picture offered by crystallography, this study reveals changes in the aromatic cage on ligand binding and suggests that reduction of the cofactor substantially alters the environment of aromatic residues presumably near the flavin. In addition, the covalently modified reduced MAO A shows significant differences from the substrate-reduced enzyme. D

a b s t r a c t 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 amethylated 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 À8 M range, with selectivities towards human MAO-B exceeding 2000-fold.

It has been accepted that, as required mechanistically, the neutral form of the amine is the substrate for monoamine oxidase, despite the amine pK a of above 9.5. The pH dependence of the kinetic parameters for kynuramine oxidation by purified human MAO-A and for phenylethylamine oxidation by MAO-B in granulocytes at pH values from 5 to 10 was consistent with the protonated amine being used. Deprotonation of a group of pK a ¼ 7.1 in MAO-B and pK a ¼ 7.5 AE 0.1 (n ¼ 4) in MAO-A was important for efficient catalysis. The K i values for two oxazolidinone inhibitors of MAO-A gave opposite pH-dependence indicating that the uncharged form of each inhibitor bound better than the charged form. Decreased pH induced a blue shift in the spectral maximum of MAO-A indicative of a more hydrophobic environment around the flavin, and also influenced the redox properties of the flavin.

Carnitine acetyltransferase (CrAT) is part of the carnitine system that protects the acylation state of the pools of acetyl-coenzyme A, a key metabolic intermediate, by transferring excess acetate and other shortchain acyl groups to and from carnitine. The homology of CrAT with other carnitine acyltransferases, such as carnitine palmitoyltransferase I (CPT-I) that regulates fatty acid metabolism, make the solving of its structure a landmark in understanding mechanism and ligand binding in this family.

Monoamine oxidases A and B have identical flavin sites but different, although overlapping, amine substrate specificity. Reoxidation of ternary complexes containing substrate is much faster than of free enzyme, and the enhancement is greater in the A form than the B form. The oxidative half-reaction was studied with a variety of substrates to elucidate the specificity of the effect and to probe the different influences of substrate on the flavin reoxidation in the two forms of the enzyme. The second-order rate constant for the reoxidation was highest with monoamine oxidase A when kynuramine was the ligand (508 x 103 M-' S-1 ) c ompared to 4 X lo3 M-' s-l in its absence. MPTP (166 X lo3 M-l s-' ) a lso enhanced reoxidation well, but indole substrates stimulated only poorly (e.g., tryptamine, 29 X lo3 M-l s-l; serotonin, 50 X lo3 M-l s-l). For the A form, the reduction of the flavin was rate-limiting in all cases. For the B , CA 94121. Telephone: 415-752-9676; fax: Abbreviations: MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; MPDEH, 1 -methyl-4-phenyl-2,3d~ydropyrid~um; MPEH, l-methyl-4-phenylpyridinium; MAO, monoamine oxidase. 415-750-6959.

Monoamine oxidases A and B have identical flavin sites but different, although overlapping, amine substrate specificity. Reoxidation of ternary complexes containing substrate is much faster than of free enzyme, and the enhancement is greater in the A form than the B form. The oxidative half-reaction was studied with a variety of substrates to elucidate the specificity of the effect and to probe the different influences of substrate on the flavin reoxidation in the two forms of the enzyme. The second-order rate constant for the reoxidation was highest with monoamine oxidase A when kynuramine was the ligand (508 x 103 M-' S-1 ) c ompared to 4 X lo3 M-' s-l in its absence. MPTP (166 X lo3 M-l s-' ) a lso enhanced reoxidation well, but indole substrates stimulated only poorly (e.g., tryptamine, 29 X lo3 M-l s-l; serotonin, 50 X lo3 M-l s-l). For the A form, the reduction of the flavin was rate-limiting in all cases. For the B , CA 94121. Telephone: 415-752-9676; fax: Abbreviations: MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine; MPDEH, 1 -methyl-4-phenyl-2,3d~ydropyrid~um; MPEH, l-methyl-4-phenylpyridinium; MAO, monoamine oxidase. 415-750-6959.

A series of pirlindole analogues were tested as inhibitors of monoamine oxidase A and B. Although we did not find strict dependence between 3D-size of molecules and their inhibitory potency, rigid analogues exhibited potent and selective inhibition of MAO-A. They have 3D size limits of 13 angstroms (length) x 7 angstroms (height) x 4.4 angstroms (widths). Besides MAO-A inhibition flexible analogues also demonstrated potent inhibition of MAO-B. Five compounds were studied as inhibitors of purified human liver MAO-A. Their inhibitory potencies coincided with those obtained using rat liver mitochondrial MAO-A. Each compound induced changes in the spectrum of MAO-A but these did not correlate with the flexibility of the derivative. It is also possible that the oxygen bridge introduced with the flexibility might influence spectral patterns.

This article summarizes recent studies in the authors' and other laboratories of selective inhibitors acting at the 'rotenone' site and at the Q binding site in the NADH-Q oxidoreductase segment of the respiratory chain. A wide array of inhibitors act at the rotenone site to block electron flux from the enzyme to the Q pool. Using evidence from studies with rotenone, piericidin A, and analogs of the neurotoxic N-methyl-4-phenylpyridinium, we have proposed two binding sites for these inhibitors, both of which must be occupied for complete inhibition of NADH oxidation.

This paper examines the experimental foundations of reports in the literature on mitochondrial diseases involving Complexes I and I| of the respiratory chain. Many of the reports may be questioned on the basis of the assay conditions used which disregard established knowledge of the precautions required for valid activity measurements. In addition, some findings are open to question because of the experimental material chosen for the study, such as the measurement of NADH oxidase activity in platelets in Parkinson's disease, which affects selectively the dopamine neurons, or the use of autopsy material stored for prolonged periods during which post-mortem changes may have occurred. Deficiencies claimed to involve several components of the respiratory chain may reflect indirect effects, such as defects in the synthesis of iron-sulfur clusters or in the availability of iron, rather than mutations in the genes coding for the deficient enzymes. Nevertheless, there are a few instances reported of Complex II deficiency free from such criticisms. As to Complex I, idiopathic Parkinsonism appears to involve a documentable decline in the activity of this enzyme. Using the model system provided by N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), which produces biochemical, pharmacological, and clinical syndromes closely resembling Parkinsonism, the etiology of the disease is examined.

Carnitine (L-3-hydroxy-4-N-trimethylaminobutyric acid) forms esters with a wide range of acyl groups and functions to transport and excrete these groups. It is found in most cells at millimolar levels after uptake via the sodium-dependent carrier, OCTN2. The acylation state of the mobile carnitine pool is linked to that of the limited and compartmentalised coenzyme A pools by the action of the family of carnitine acyltransferases and the mitochondrial membrane transporter, CACT. The genes and sequences of the carriers and the acyltransferases are reviewed along with mutations that affect activity. After summarising the accepted enzymatic background, recent molecular studies on the carnitine acyltransferases are described to provide a picture of the role and function of these freely reversible enzymes. The kinetic and chemical mechanisms are also discussed in relation to the different inhibitors under study for their potential to control diseases of lipid metabolism. ß

In this paper we report that palmitoyl-L-carnitine can be a metabolic intermediate of the fatty acid incorporation pathway into erythrocyte membrane phosphatidylcholine, and phosphatidylethanolamine. Phospholipid acylation was evaluated by measuring the incorporation of radioactive [1-14C]-palmitoyl-L-carnitine in membrane erythrocyte ghost phospholipids in the presence or absence of CoA. CoA highly stimulated the incorporation of [ 1-14C]-palmitic acid into both the phospholipids examined, although the incorporation was also evident in the absence of added CoA. Incorporation of [1-14C]-palmitic acid into phosphatidylcholine was greater than into phosphatidylethanolamine. 2-Bromo-palmitoyl-CoA, an irreversible inhibitor of the erythrocyte carnitine palmitoyltransfemse, inhibited the acylation process.