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We have structure, a wealth of kinetic data, thousands of chemical ligands and clinical information for the effects of a range of drugs on monoamine oxidase activity in vivo. We have comparative information from various species and mutations on kinetics and effects of inhibition. Nevertheless, there are what seem like simple questions still to be answered. This article presents a brief summary of existing experimental evidence the background and poses questions that remain intriguing for chemists and biochemists researching the chemical enzymology of and drug design for monoamine oxidases (FAD-containing EC 4.1.3.4).

The covalently bound FAD cofactor in monoamine oxidase (MAO) is reduced by the amine substrate and reoxidized by oxygen. Visible spectroscopy provides a convenient tool to study the interaction of ligands and the kinetics of the half-reactions for mechanistic investigations. Equilibrium redox titrations allow measurement of redox potentials, while rapid mixing experiments allow determination of the rate of reduction by different substrates and of covalent adduct formation by irreversible inactivators. Three techniques are described: (1) measuring ligand interactions by alterations in the spectrum, especially at 495 nm; (2) reducing MAO, including the essentials for anaerobic procedures; and (3) studying kinetics of reduction, reoxidation, or inactivation of MAO.

Authors would like to acknowledge ARUK for supporting this research. YC is Chinese Scholarship recipient. The University of St Andrews is a charity registered in Scotland: No SC013532 Mitochondrial dysfunction has a recognised role in the progression of Alzheimer's disease (AD) pathophysiology. Cerebral perfusion becomes increasingly inefficient throughout ageing, leading to unbalanced mitochondrial dynamics. This effect is exaggerated by amyloid β (Aβ) and phosphorylated tau, two hallmark proteins of AD pathology. A neuroprotective role for the adipose‐derived hormone, leptin, has been demonstrated in neuronal cells. However, its effects with relation to mitochondrial function in AD remain largely unknown. To address this question, we have used both a glucose‐serum deprived (CGSD) model of ischaemic stroke in SH‐SY5Y cells and a Aβ1‐42‐treatment model of AD in differentiated hippocampal cells. Using a combination of JC‐1 and MitoRed staining techniques, we show that leptin prevents depolarisation of the mitochondrial membrane and excessive mitochondrial fragmentation induced by both CGSD and Aβ1‐42. Thereafter, we used ELISAs and a number of activity assays to reveal the biochemical underpinnings of these processes. Specifically, leptin was seen to inhibit upregulation of the mitochondrial fission protein Fis1 and downregulation of the mitochondrial fusion protein, Mfn2. Furthermore, leptin was seen to upregulate the expression and activity of the antioxidant enzyme, monoamine oxidase B. Herein we provide the first demonstration that leptin is sufficient to protect against aberrant mitochondrial dynamics and resulting loss of function induced by both CGSD and Aβ1‐42. We conclude that the established neuroprotective actions of leptin may be facilitated through regulation of mitochondrial dynamics. Publisher PDF

Based on the monoamine oxidase (MAO) inhibition properties of aminoheterocycles with a carbonitrile group we have carried out a systematic exploration to discover new classes of carbonitriles endowed with dual MAO and AChE inhibitory activities, and Aβ anti-aggregating properties. Eighty-three nitrile-containing compounds, 13 of which are new, were synthesized and evaluated. in vitro screening revealed that 31, a new compound, presented the best lead for trifunctional inhibition against MAO A (0.34 μM), MAO B (0.26 μM), and AChE (52 μM), while 32 exhibited a lead for selective MAO A (0.12 μM) inhibition coupled to AChE (48 μM) inhibition. Computational analysis revealed that the malononitrile group can find an advantageous position with the aromatic cleft and FAD of MAO A or MAO B. However, the total binding energy can be handicapped by an internal penalty caused by twisting of the ligand molecule and subsequent disruption of the conjugation (32 in MAO B compared to the conjugated 31). Conjugation is also important for AChE as well as the hydrophilic character of malononitrile that allows this group to be in close contact with the aqueous environment as seen for 83. Although the effect of 31 and 32 against Aβ, was very weak, the effect of 63 and 65, and of the new compound 75, indicated that these compounds were able to disaggregate Aβ fibrils. The most effective was 63, a (phenylhydrazinylidene)propanedinitrile derivative that also inhibited MAO A (1.65 μM), making it a potential lead for Alzheimer's disease application., European Cooperation in Science and Technology, Grant/Award Numbers: STSM CM1103-29512, STSM D34-05989, STSMCM1103- 29510; Portuguese Foundation for Science and Technology, Grant/Award Number: FCT -Project PTDC/SAUNEU/ 64151/2006 (MCC); Slovak Research and Development Agency (MM); Slovak Research and Development Agency; Foundation for Science and Technology

The extensive research conducted on monoaminergic systems including dopaminergic, serotonergic, noradrenergic, histaminergic and, to a lower extent melatonergic systems, continues to expand our current knowledge. The topic has enormous health, economic, scientific and conceptual stakes, with engagement across disciplines covering medicinal chemistry to neuropsychiatric and neurological diseases. This special issue presents scientific updates with in depth analyses on selected aspects of this complicated topic., peer-reviewed

This publication was supported by the project Czech National Institute of Mental Health (NIMH – CZ; no. ED2.1.00/03.0078), Ministry of Education, Youth and Sports of the Czech Republic (no. LD14009), MH CZ - DRO (UHHK, 00179906), the University of Defence of the Czech Republic (long term development plan), MINECO (Government of Spain; Grant SAF2012-33304), the School of Biology at the University of St Andrews, and by COST Action CM1103. In this study, novel indolotacrine analogues have been designed, synthesized and evaluated as potential drugs for the treatment of Alzheimer’s disease. Based on a multi-target-directed ligand approach, novel compounds have been designed to act simultaneously as cholinesterase and monoamine oxidase (MAO) inhibitors. Prepared compounds were also evaluated for their antioxidant, cytotoxic, hepatotoxic and permeability (Blood-Brain Barrier penetration) properties. Indolotacrine 9b (9-methoxy-2,3,4,6-tetrahydro-1H-indolo[2,3-b]quinolin-11-amine) showed the most promising results in the in vitro assessment being a potent inhibitor of acetylcholinesterase (IC50 = 1.5 µM), butyrylcholinesterase (IC50 = 2.4 µM) and monoamine oxidase A (IC50 = 0.49 µM) and a weak inhibitor of monoamine oxidase B (IC50 = 53.9 µM). Although its cytotoxic (IC50 = 5.5 ± 0.4 µM) and hepatotoxic (IC50 = 1.22 ± 0.11 µM) profile is not as good as the standard 7-methoxytacrine (IC50 = 63 ± 4 µM and IC50 = 11.50 ± 0.77 µM respectively), the overall improvement in the inhibitory activities and potential to cross blood-brain barrier make indolotacrine 9b a promising lead compound for further development and investigation. Postprint

We thank our funding sources: COST Action CM1103 STSM14325 and the School of Biology at the University of St Andrews to TM; Ghent University (BOF) and the Research Foundation - Flanders (FWO - Vlaanderen) to SM; and the University of East Anglia (AG). Cyclopropylamines, inhibitors of monoamine oxidases (MAO) and lysine-specific demethylase (LSD1), provide a useful structural scaffold for the design of mechanism-based inhibitors for the therapy of depression and cancer. For new compounds with the less common cis relationship and with an alkoxy substituent at the 2-position of the cyclopropyl ring, the apparent affinity from docking revealed little difference between the enantiomers. Using the racemate, kinetic parameters for the reversible and irreversible inhibition of MAO were determined. No inhibition of LSD1 was observed. The reversible inhibition of MAO A gave high IC50 values for most compounds but sub-micromolar values with MAO B. After pre-incubation with the enzyme, the inhibition was irreversible in both MAO A and MAO B and the activity was not restored by dilution. Spectral changes during inactivation of MAO A showed bleaching at 456 nm and an increase at 400 nm, consistent with flavin modification. These derivatives are MAO B-selective mechanism-based inhibitors without inhibition of LSD1. The best inhibitor was cis-N-benzyl-2-methoxycyclopropylamine, with an IC50 of 5 nM for MAO B and 170 nM for MAO A after 30 min preincubation. This cis-cyclopropylamine is over 20-fold more effective than tranylcypromine so could be studied as a lead for selective inhibitors of MAO B that do not inhibit LSD1. Postprint

Monoamine oxidases (MAOs) catalyse the oxidation of neurotransmitter amines and a wide variety of primary, secondary and tertiary amine xenobiotics, including therapeutic drugs. While inhibition of MAO activity in the periphery removes protection from biogenic amines and so is undesirable, inhibition in the brain gives vital antidepressant and behavioural advantages that make MAO a major pharmaceutical target for inhibitor design. In neurodegenerative diseases, MAO inhibitors can help to maintain neurotransmitter levels, making it a common feature in novel multi-target combinations designed to combat Alzheimer's disease, albeit not yet proven clinically. Vital information for inhibitor design comes from an understanding of the structure, mechanism, and kinetics of the catalyst. This review will summarize the kinetic behaviour of MAO A and B and the kinetic evaluation of reversible inhibitors that transiently decrease catalysis. Kinetic parameters and crystal structures have enabled computational approaches to ligand discovery and validation of hits by docking. Kinetics and a wide variety of substrates and inhibitors along with theoretical modelling have also contributed to proposed schemes for the still debated chemical mechanism of amine oxidation. However, most of the marketed MAO drugs are long-lasting irreversible inactivators. The mechanism of irreversible inhibition by hydrazine, cyclopropylamine, and propargylamine drugs will be discussed. The article finishes with some examples of the propargylamine moiety in multi-target ligand design to combat neurodegeneration.

The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC50 value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behaviour remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.

The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC50 value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behaviour remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.

The therapy of complex neurodegenerative diseases requires the development of multitarget-directed drugs (MTDs). Novel indole derivatives with inhibitory activity towards acetyl/butyrylcholinesterases and monoamine oxidases A/B as well as the histamine H

The regulation of brain monoamine levels is paramount for cognitive functions, and the monoamine oxidase (MAO A and B) enzymes play a central role in these processes. The aim of this study was to evaluate whether the procognitive properties exerted by propargylamine N-(furan-2-ylmethyl)-N-methylprop-2-yn-1-amine (F2MPA) are related to changes in monoamine content via MAO inhibition. In vivo microdialysis and ex vivo amine metabolite measurement demonstrated region-specific alterations in monoamine metabolism that differ from both of the classic MAO A and MAO B inhibitors, clorgyline and l-deprenyl, respectively. Although all the inhibitors (1 and 4 mg/kg) increased cortical serotonin tissue content, only F2MPA increased the levels of cortical noradrenaline. In the striatum, clorgyline (1 mg/kg), but not F2MPA (1 mg/kg), reduced extracellular levels of dopamine metabolites at rest or stimulated by the intrastriatal application of the MAO substrate 3-methoxytyramine. In vitro, F2MPA exhibited a low affinity toward MAO B and MAO A. Nonetheless, it modified the B form of MAO, forming a flavin adduct structurally similar to that with deprenyl. F2MPA was rapidly metabolized in the presence of rat but not human microsomes, producing a hydroxylated derivative. In conclusion, the effect of F2MPA on cognition may arise from monoaminergic changes in the cortex, but the role of MAO in this process is likely to be negligible, consistent with the poor affinity of F2MPA for MAO., Collaborations among authors were enabled by COST Action CM1103, Structure-based drug design for diagnosis and treatment of neurological diseases. C.B. thanks Fondazione Cariplo (project no. 2014-0672) and the BioStruct-X program (project no. 10205) for funding synchrotron trips. P.D.D. thanks the support of Idex Bordeaux (PEPS IDEX project 2013).

The therapy of complex neurodegenerative diseases requires the development of multitarget-directed drugs (MTDs). Novel indole derivatives with inhibitory activity towards acetyl/butyrylcholinesterases and monoamine oxidases A/B as well as the histamine H receptor (H3R) were obtained by optimization of the neuroprotectant ASS234 by incorporating generally accepted H3R pharmacophore motifs. These small-molecule hits demonstrated balanced activities at the targets, mostly in the nanomolar concentration range. Additional in vitro studies showed antioxidative neuroprotective effects as well as the ability to penetrate the blood–brain barrier. With this promising in vitro profile, contilisant (at 1 mg kg i.p.) also significantly improved lipopolysaccharide-induced cognitive deficits., J.M.C. thanks MINECO (SAF2012-33304 and SAF2015- 65586-R). J.M.C., F.L.M., and A.R. thank UCJC for grants 2015-12, 2014-35, and 2015-21, respectively. J.E. thanks the Fondo de Investigaciones Sanitarias (FIS) (ISCIII/FEDER) (Programa Miguel Servet: CP14/00008 and PI16/00735) and Fundación Mutua Madrileña. O.S. and J.J. thank MHCZDRO (UHHK 00179906) for support. R.R.R., H.S., and J.M.C. acknowledge the EU COST Actions CM1103 and CM15135. E.P. and H.S. thank the German Research Foundation (DFG; PRO 1405/2-2, PRO 1405/4-1, SFB 1039 A07, and INST208/664-1).

Monoamine oxidase (MAO, EC.1.4.3.4) has been a drug target for 60 years, with the primary rationale of developing drugs to treat neuropsychiatric disorders. The biological importance of MAO is to regulate amine levels is the brain and to metabolize amines and drugs in the periphery. This review of the biochemistry of MAO A and MAO B describes the functional properties of the two enzymes integrated with knowledge of the structures of the many MAOinhibitor complexes published in the last 10 years. The analysis of activity, and the chemical and kinetic mechanisms are discussed. Inhibition studies on human MAO in vitro are now facilitated by assays using readily available materials but the kinetics of MAO involving alternative oxidative pathways and sensitivity to the oxygen concentration mean that careful analysis of the data is required as well as the good practice of determining mechanism of inhibition and kinetic constants. Kinetic constants can then be compared with thermodynamic calculations. Inhibitors bind to both the oxidized and reduced forms of MAO present during turnover so both forms should be considered when using molecular dynamics to facilitate drug design. Interaction of inhibitors with the active site can be detected as changes in the visible spectrum and these changes can provide clues about the flavin adduct formed for irreversible inhibitors or about the proximity to the flavin for reversible inhibitors. Developing areas (knock-out mice for behavior, the imidazoline binding site, and imaging to monitor the activity and the inhibition of MAO in the patient) are mentioned.

HIGHLIGHTS • Compounds that interact with multiple targets but minimally with the cytochrome P450 system (CYP) address the many factors leading to neurodegeneration. • Acetyl- and Butyryl-cholineEsterases (AChE, BChE) and Monoamine Oxidases A/B (MAO A, MAO B) are targets for Multi-Target Designed Ligands (MTDL). • ASS234 is an irreversible inhibitor of MAO A > MAO B and has micromolar potency against the cholinesterases. • ASS234 is a poor CYP substrate in human liver, yielding the depropargylated metabolite. • SMe1EC2, a stobadine derivative, showed high radical scavenging property, in vitro and in vivo giving protection in head trauma and diabetic damage of endothelium. • Control of mitochondrial function and morphology by manipulating fission and fusion is emerging as a target area for therapeutic strategies to decrease the pathological outcome of neurodegenerative diseases. Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to "dirty drugs" for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson''s Disease''s (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are simply symptomatic treatment so if new drugs are to prevent degeneration rather than compensate for loss of neurotransmitters, then oxidative stress and mitochondrial events must also be targeted. MAO inhibitors can protect neurons from apoptosis by mechanisms unrelated to enzyme inhibition. Understanding the involvement of MAO and other proteins in the induction and regulation of the apoptosis in mitochondria will aid progress toward strategies to prevent the loss of neurons. In general, the oxidative stress observed both in PD and AD indicate that antioxidant properties are a desirable part of MTDL molecules. After two or more properties are incorporated into one molecule, the passage from a lead compound to a therapeutic tool is strictly linked to its pharmacokinetic and toxicity. In this context the interaction of any new molecules with cytochrome P450 and other xenobiotic metabolic processes is a crucial point. The present review covers the biochemistry of enzymes targeted in the design of drugs against neurodegeneration and the cytochrome P450-dependent metabolism of MTDLs.

HIGHLIGHTS Many AD target combinations are being explored for multi-target drug design. New databases and models increase the potential of computational drug design Liraglutide and other antidiabetics are strong candidates for repurposing to AD. Donecopride a dual 5-HT/AChE inhibitor shows promise in pre-clinical studies Alzheimer's Disease is a complex and multifactorial disease for which the mechanism is still not fully understood. As new insights into disease progression are discovered, new drugs must be designed to target those aspects of the disease that cause neuronal damage rather than just the symptoms currently addressed by single target drugs. It is becoming possible to target several aspects of the disease pathology at once using multi-target drugs (MTDs). Intended as an introduction for non-experts, this review describes the key MTD design approaches, namely structure-based, in silico, and data-mining, to evaluate what is preventing compounds progressing through the clinic to the market. Repurposing current drugs using their off-target effects reduces the cost of development, time to launch, and the uncertainty associated with safety and pharmacokinetics. The most promising drugs currently being investigated for repurposing to Alzheimer's Disease are rasagiline, originally developed for the treatment of Parkinson's Disease, and liraglutide, an antidiabetic. Rational drug design can combine pharmacophores of multiple drugs, systematically change functional groups, and rank them by virtual screening. Hits confirmed experimentally are rationally modified to generate an effective multi-potent lead compound. Examples from this approach are ASS234 with properties similar to rasagiline, and donecopride, a hybrid of an acetylcholinesterase inhibitor and a 5-HT4 receptor agonist with pro-cognitive effects. Exploiting these interdisciplinary approaches, public-private collaborative lead factories promise faster delivery of new drugs to the clinic.

The search for new monoamine oxidase inhibitors aims to identify potential lead compounds that are more potent and selective than current drugs for use in treating a variety of neuropsychiatric and neurodegenerative conditions. An integral part of this process is a kinetic examination of monoamine oxidases in the presence of the inhibitor, to determine potency and selectivity and to obtain information on mechanism. To date, kinetic data obtained with a probe substrate have been analysed by fitting to the Michaelis-Menten equation which describes a unireactant process in which velocity is related to substrate concentration in a rectangular hyperbolic manner. In this study, we present evidence that monoamine oxidase activity is often not adequately described by this approach. We outline a novel equation strategy that takes account of substrate and inhibitor binding to oxidised and reduced enzyme forms, and quantifies differences between substrates and inhibitors in this regard. When combined with plate reader-based experimental techniques that allow large numbers of substrate and inhibitor concentrations to be used, and the global nonlinear regression facilities of GraphPad Prism software, this straightforward approach allows more appropriate analyses of monoamine oxidase by non-experts than has previously been possible.

Brain monoamine neurotransmitters, which include dopamine (DA), noradrenaline (NA), adrenaline (A) serotonin (5-HT) histamine, and melatonin, play a central role in the pathophysiology of all major neuropsychiatric disorders, such as mood disorders, schizophrenia, autism-spectrum disorders, Parkinson disease (PD), epilepsy, and dementias. Ligands for the receptors acted upon by the monoamine neurotransmitters and inhibitors of the enzymes for breakdown, and the reuptake transporters have been developed as drugs, but successful therapy of neuropsychiatric disorders is limited by the high variability of the symptoms and of the responses to treatment. Few drugs are available to address specific subsets of neurological/mental symptoms and none to aid in diagnosis or to stop the progress of neurodegenerative disorders., peer-reviewed

An allosteric binding site with high affinity for imidazoline I 2 ligands has been proposed to exist on monoamine oxidase-B (MAO-B). However, enzyme inhibition only occurs at ligand concentrations far higher than are required to saturate this site. We here confirm previous reports that inactivation of recombinant human MAO-B with tranylcypromine results in the formation of a high affinity I 2 site on the enzyme, measured as an increase in binding of [ 3 H]2-BFI. Incubation of MAO-B with 2-phenylethylamine, an endogenous trace amine and MAO-B substrate, resulted in a progressive loss of enzyme activity, increased enzyme mass, distinct spectral changes and, as was observed with tranylcypromine, a parallel increase in high affinity binding of [ 3 H]2-BFI. Kinetic studies of the mechanism by which 2-BFI inhibits MAO-B activity suggested binding of 2-BFI, at micromolar concentrations, to a site distinct from the active site on at least two forms of the pure enzyme, probably corresponding to oxidised and reduced enzyme states. Studies with mutant enzymes revealed a pattern of changes consistent with binding of 2-BFI to the substrate entrance channel of human MAO-B. Structural data confirm that high affinity binding of I 2 ligands occurs within the entrance channel of inactive enzyme, while lower affinity binding at the same location in catalytically active enzyme results in mixed inhibition of MAO-B activity. High affinity I 2 sites may form in vivo due to inactivation of a portion of MAO-B during amine oxidation, while the low affinity I 2 site on active enzyme is a target for novel MAO-B inhibitor drugs.

A variety of approaches have been taken to improve the brain penetration of pharmaceutical agents. The amphipathic character of a compound can improve its interaction with the lipid bilayer within cell membranes, and as a result improve permeability. Fatty acid chains or lipoamino acids of various lengths were attached to tranylcypromine (TCP), in an attempt to improve the blood-brain barrier (BBB) permeability by increasing the lipophilicity as well as the amphiphatic character of the drug. TCP-FA4, one of the derivatives containing a four carbon alkyl acid chain, showed the greatest improvement in permeability. This molecule was slightly neuroprotective in a beta-amyloid-induced neurodegeneration assay and may also be capable of upregulating brain derived neurotrophic factor (BDNF), as indicated by cell culture assays using human umbilical vein endothelial cells. Since decreased levels of BDNF are observed in many CNS disorders, and direct injection of BDNF is not a viable option due to its poor permeability across the BBB, small molecules capable of regulating BDNF that also cross the BBB may be an interesting treatment option.

Carnitine is important for cell function and survival primarily because of its involvement in the multiple equilibria between acylcarnitine and acyl-CoA esters established through the enzymatic activities of the family of carnitine acyltransferases. These have different acyl chain-length specificities and intracellular compartment distributions, and act in synchrony to regulate multiple aspects of metabolism, ranging from fuel-selection and -sensing, to the modulation of the signal transduction mechanisms involved in many homeostatic systems. This review aims to rationalise the extensive range of experimental and clinical data that have been obtained through the pharmacological use of L-carnitine and its short-chain acylesters, over the past two decades, in terms of the basic biochemical mechanisms involved in the effects of carnitine on the various cellular acyl-CoA pools in health and disease.

Background and purpose: Monoamine oxidase inhibitors (MAOI) are known to cause serotonin toxicity (ST) when administered with selective serotonin reuptake inhibitors (SSRI). Methylene blue (methylthionium chloride, MB), a redox dye in clinical use, has been reported to precipitate ST in patients using SSRI. MB was assessed for MAO inhibition and so for its potential to precipitate ST. Experimental approach: Inhibition of purified human MAO was quantified using kinetic assays and visible spectral changes to study the interactions of MB with MAO A. Key results: MB was a potent (tight binding) inhibitor for MAO A. It also inhibited MAO B but at much higher concentration. Interactions of MB with the active site of MAO A were confirmed by its action both as an oxidising substrate and as a one-electron reductant. Conclusions and implications: MB is a potent reversible inhibitor of MAO A with implications for gut uptake of amines when administered orally. At concentrations reported in the literature after intravenous administration, MAO B would be partially inhibited but MAO A would be completely inhibited. This inhibition of MAO A would be expected to lead to perturbations of 5-hydroxytryptamine metabolism and hence account for ST occurring when administered to patients on SSRI treatment. British Journal of Pharmacology (2007) 152, 946–951; doi:10.1038/sj.bjp.0707430; published online 27 August 2007

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) = 7.1 in MAO-B and pK (a) = 7.5 +/- 0.1 (n = 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.

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & Garcia-Sevilla JA (1997) Br J Pharmacol121, 901-912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K(i) values of 3 microM, 26 microM and 125 microM, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 +/- 3.9 microM) and 2-(2-benzofuranyl)-2-imidazoline (49 +/- 8 microM). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.

Recently developed multi-targeted ligands are novel drug candidates able to interact with monoamine oxidase A and B; acetylcholinesterase and butyrylcholinesterase; or with histamine N-methyltransferase and histamine H3-receptor (H3R). These proteins are drug targets in the treatment of depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease. A probabilistic method, the Parzen-Rosenblatt window approach, was used to build a >predictor> model using data collected from the ChEMBL database. The model can be used to predict both the primary pharmaceutical target and off-targets of a compound based on its structure. Molecular structures were represented based on the circular fingerprint methodology. The same approach was used to build a >predictor> model from the DrugBank dataset to determine the main pharmacological groups of the compound. The study of off-target interactions is now recognised as crucial to the understanding of both drug action and toxicology. Primary pharmaceutical targets and off-targets for the novel multi-target ligands were examined by use of the developed cheminformatic method. Several multi-target ligands were selected for further study, as compounds with possible additional beneficial pharmacological activities. The cheminformatic targets identifications were in agreement with four 3D-QSAR (H3R/D1R/D2R/5-HT2aR) models and by in vitro assays for serotonin 5-HT1a and 5-HT2a receptor binding of the most promising ligand (71/MBA-VEG8).

The structure of carnitine acetyltransferase revealed a putative binding site for longer acyl chains but access was blocked by methionine 564 (G. Jogl and L. Tong (2003) Cell 112, 113–122). The equivalent residue in all long chain carnitine acyltransferases is a conserved glycine. Mutation of glycine 553 to methionine in bovine COT resulted in loss of activity with all acyl-CoA substrates except acetyl-CoA, supporting the hypothesis that the methionine blocks access for longer acyl chains. The kinetic characteristics of acetyl transfer to carnitine were identical in the native and mutant enzyme. However, rapid acetyl-CoA hydrolysis in the mutant but not the wild-type indicates perturbation of the catalytic site.

Oxazolidinones represent a new and promising class of antibacterial agents. Current research in this area is mainly concentrated on improving the safety profile and the antibacterial spectrum. Many oxazolidinones, including linezolid (marketed as Zyvox), are inhibitors of monoamine oxidase A (MAO-A), which presents an undesired side effect. Recently, it was found that the 1,2,3-triazole is a good replacement for the conventional acetamide functionality found in oxazolidinones. We now disclose the finding that 1,2,3-triazoles bearing a substituent like methyl, small substituted methyl, bromo, or a linear (sp-hybridized) group at the 4 position (compounds such as 5, 16, 19, and 21) are good antibacterials with reduced or no activity, within the detection limit of the assay, against MAO-A. The results are especially promising for the development of oxazolidinones with an improved safety profile. The MAO-A SAR can be rationalized on the basis of docking studies to a MAO-A/MAO-B homology model.

Cells contain limited and sequestered pools of Coenzyme A (CoA) that are essential for activating carboxylate metabolites. Some acyl-CoA esters have high metabolic and signalling impact, so control of CoA ester concentrations is important. This and transfer of the activated acyl moieties between cell compartments without wasting energy on futile cycles of hydrolysis and resynthesis is achieved through the carnitine system. The location, properties of and deficiencies in the carnitine acyltransferases are described in relation to their influence on the CoA pools in the cell and, hence, on metabolism. The protection of free CoA pools in disease states is achieved by excretion of acyl-carnitine so that carnitine supplementation is required where unwanted acyl groups build up, such as in some inherited disorders of fatty acid oxidation. Acetyl-carnitine improves cognition in the brain and propionyl-carnitine improves cardiac performance in heart disease and diabetes. The therapeutic effects of carnitine and its esters are discussed in relation to the integrative influence of the carnitine system across CoA pools. Recent evidence for sequestered pools of activated acetate for synthesis of malonyl-CoA, for the synthesis of polyunsaturated fatty acids and for the inhibition of carnitine palmitoyltransferase 1 to regulate fatty acid oxidation is reviewed.

The structure of monoamine oxidase B 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-8-methyl-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.

This special issue, “Monoamine oxidase (MAO) and aggression”, considers the evidence linking the monoaminergic neurotransmitters as regulated by the breakdown enzymes MAO-A and MAO-B, and aggressive behavior. This issue of Progress in Neuro-Psychopharmacology and Biological Psychiatry presents five reviews and one original research article from the experts in the field. All articles offer comprehensive analyses and novel findings on the role of MAO in aggression. The common link for all articles is that they incorporate basic and clinical research and connect data from psychiatry, neurology, psychology, neuropharmacology and medicinal chemistry. These articles explore the association between MAO abnormalities and aggressive behavior, possible mechanisms underlying vulnerability to environmental adversity in the pathogenesis of aggression, as well as therapeutic strategies focusing on MAO targets. Encoded by separate genes on the X chromosome, MAO-A and B both catalyze the breakdown of amines but have different catalytic properties, distinctly different distribution across the brain and diverse effects on various personality traits and behaviors.

Reversible monoamine oxidase A inhibitors (RIMA) are used as antidepressants but little is known about how they interact with the active site of the enzyme. Heterologous expression of human liver MAO-A in yeast provides sufficient protein for molecular studies and direct observation of the changes in the spectrum of the FAD co-factor when inhibitors bind. Using the reversible inhibitor, D-amphetamine, as a model compound, a concentration-dependent change in the spectrum with clean isosbestic points was observed. The decrease in absorbance between 400 and 500 nm gave a dissociation constant for binding similar to the K(i) value. Anaerobic reduction yielded the semiquinone spectrum only and the midpoint potential was the same as the free enzyme. Full reduction was not possible with dithionite as the reductant, suggesting that the semiquinone-reduced couple had a much lower midpoint potential than the free enzyme. In contrast, with substrate, which reduces the enzyme on an equimolar basis, the semiquinone is never seen. In anaerobic stopped-flow experiments, amphetamine inhibits completely the reoxidation of the reduced enzyme in contrast to a substrate such as 2-phenylethylamine (the desmethyl analogue of amphetamine) that accelerates the rate 12-fold. The spectral changes in MAO-A permit the examination of inhibitor interaction with the redox co-factor. Stacking of the inhibitor and flavin rings constitutes part of the interaction but, taking into account other evidence, steric factors may be the clue to the differences between substrate and inhibitor.

Monoamine oxidase (MAO) inhibitors were developed as antidepressants but many drugs, including the novel oxazolidinone antibacterial agents, share similar molecular properties and have MAO inhibitory activity. Factors important for binding antidepressants and modifications to decrease binding of oxazolidinones to avoid undesirable vascular effects are discussed.

1-Methyl-4-phenylpyridinium (MPP+), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4'-alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 microM and from 1.6 to 3,300 microM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4'-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 microM in mitochondria and from 1.7 to 142 microM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an approximately 600-fold, versus only a 36-fold, increase in the IC50 of MPP+ versus 4'-pentyl-MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4'-decyl-MPP+ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol.(ABSTRACT TRUNCATED AT 250 WORDS)

In higher eukaryotes three different VDAC genes encode three homologous proteins which do not show the same activity. VDAC1 and VDAC2 isoforms have been characterized while VDAC3 isoform is still elusive. To explore VDAC3 protein interactions, we have established a stable cell line expressing a fluorescent and dual-tagged construct. This clone expresses a stable amount of VDAC3. Live cell imaging shows that fluorescent VDAC3 localizes in the mitochondria. Proteins interacting with VDAC3 have been separated by tandem-affinity purification and 2-D gel electrophoresis and identified by mass spectrometry. In the list of putative interacting proteins, there are cytosolic, mitochondrial, cytoskeletal and ER proteins. Coherent pathways like cell redox homeostasis, response to stress, formation/rearrangement of disulfide bonds, response to unfolded proteins or protein folding have been found to be related to clusters of proteins identified in this experiment. The list of associated proteins has been validated by immunoprecipitation experiments utilizing specific antibodies. Likely biological and pathological processes have been analyzed. Cytosolic proteins associated with VDAC3 include tubulins and cytoskeletal proteins, stress sensors, chaperones and proteasome components, redox-mediating enzymes such as protein disulphide isomerase. The overall picture points to a role for VDAC3 as mediator for the organization of protein complexes and regulator of the traffic of misfolded or non-folded proteins evoked from different stimuli.

Imidazoline ligands in I2-type binding sites in the brain alter monoamine turnover and release. One example of an I2 binding site characterized by binding studies, kinetics, and crystal structure has been described in monoamine oxidase B (MAO B). MAO A also binds imidazolines but has a different active site structure. Docking and molecular dynamics were used to explore how 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) binds to MAO A and to explain why tranylcypromine increases tight binding to MAO B. The energy for 2-BFI binding to MAO A was comparable to that for tranylcypromine-modified MAO B, but the location of 2-BFI in the MAO A could be anywhere in the monopartite substrate cavity. Binding to the tranylcypromine-modified MAO B was with high affinity and in the entrance cavity as in the crystal structure, but the energies of interaction with the native MAO B were less favorable. Molecular dynamics revealed that the entrance cavity of MAO B after tranylcypromine modification is both smaller and less flexible. This change in the presence of tranylcypromine may be responsible for the greater affinity of tranylcypromine-modified MAO B for imidazoline ligands.

Monoamine oxidases (MAO) and cholinesterases are validated targets in the design of drugs for the treatment of Alzheimer's disease. The multi-target compound N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl) methyl)-N-methylprop-2-yn-1-amine (ASS234), bearing the MAO-inhibiting propargyl group attached to a donepezil moiety that inhibits cholinesterases, retained activity against human acetyl- and butyryl-cholinesterases. The inhibition of MAO A and MAO B by ASS234 was characterized and compared to other known MAO inhibitors. ASS234 was almost as effective as clorgyline (kinact/ KI = 3 × 106 min- 1 M- 1) and was shown by structural studies to form the same N5 covalent adduct with the FAD cofactor.

The midpoint potentials for the reduction of the cysteinyl-flavin adenine dinucleotide (FAD) in monoamine oxidases (MAO) A and B in the absence and presence of ligands have been determined. Both MAO A and MAO B can be reduced chemically in two steps, the first generating a semiquinone spectrum and the second the spectrum of fully reduced FAD, each of which requires two electron equivalents. The midpoint potentials for the oxidized/semiquinone and semiquinone/reduced couples were -159+/-4 mV and -262+/-3 mV for MAO A and -167+/-4 mV and -275+/-3 mV for MAO B. After modification with a thiol reagent, direct reduction from the oxidized to fully reduced form was observed with no semiquinone and without change in the overall midpoint potential. In the presence of substrate, no semiquinone was formed, but the midpoint potential for full reduction of the flavin was positively shifted by up to 500 mV, depending on the substrate. This shift in potential could permit a more thermodynamically favorable transfer of electrons from the amine substrates to oxygen. In contrast, stable products and inhibitors did not cause a shift in potential and did not prevent the formation of semiquinone.

Peroxisomes are small, subcellular organelles that play a major role in lipid metabolism. Inherited disorders of peroxisomal structure and metabolism can result from defective assembly, missing protein import transporters, or individual enzyme deficiencies. Molecular studies helped by the range of disorders have now elucidated many of the pathways, including the paths of alpha-oxidation for phytanic acid and beta-oxidation for very-long-chain and branched-chain fatty acids and for bile acid synthesis. The mechanism of the transfer of substrates, intermediates, and products across the membrane is poorly understood. The carnitine system, known to transport activated acyl groups between localized coenzyme A pools, is presented. The evidence for the involvement of carnitine in the transfer of activated acyl groups to and from the peroxisomes is reviewed.

Since cyanide potentiates the inhibitory activity of several monoamine oxidase (MAO) inhibitors, a series of carbonitrile-containing aminoheterocycles was examined to explore the role of nitriles in determining the inhibitory activity against MAO. Dicarbonitrile aminofurans were found to be potent, selective inhibitors against MAO A. The origin of the MAO A selectivity was identified by combining spectroscopic and computational methods. Spectroscopic changes induced in MAO A by mono- and dicarbonitrile inhibitors were different, providing experimental evidence for distinct binding modes to the enzyme. Similar differences were also found between the binding of dicarbonitrile compounds to MAO A and to MAO B. Stabilization of the flavin anionic semiquinone by monocarbonitrile compounds, but destabilization by dicarbonitriles, provided further support to the distinct binding modes of these compounds and their interaction with the flavin ring. Molecular modeling studies supported the role played by the nitrile and amino groups in anchoring the inhibitor to the binding cavity. In particular, the results highlight the role of Asn181 and Ile335 in assisting the interaction of the nitrile-containing aminofuran ring. The network of interactions afforded by the specific attachment of these functional groups provides useful guidelines for the design of selective, reversible MAO A inhibitors. © 2013 Elsevier B.V.

The structural and catalytic properties of beef heart succinate dehydrogenase (succinate-ubiquinone oxidoreductase, complex II) and Escherichia coli fumarate reductase are remarkably similar. One exception is that whereas electron exchange between the mammalian enzyme and its quinone pool is inhibited by thenoyltrifluoroacetone and carboxanilides, the enzyme from E. coli is not sensitive to these inhibitors. The lack of good inhibitors has seriously hampered the elucidation of the mechanism of quinone oxidation/reduction in the E. coli enzyme. We have previously reported (Tan, A. K., Ramsay, R. R., Singer, T. P., and Miyoshi, H. (1993) J. Biol. Chem. 268, 19328-19333) that 2-alkyl-4,6-dinitrophenols inhibit mammalian complexes I, II, and III, but with different potencies and kinetic characteristics. Based on these studies we have selected a series of 2-alkyl-4,6-dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, particularly when acting in the direction of quinone reduction, the physiological event. These compounds turned out to be even more potent inhibitors of E. coli fumarate reductase, particularly when acting in the direction of quinol oxidation, again, the physiological event. Kinetic analysis revealed that with both enzymes 2 inhibitor binding sites seem to be involved in the oxidation of succinate by quinone, but one seems to be functioning when fumarate is reduced by external quinol. Since the E. coli enzyme can be modified by site-directed mutagenesis, these studies were extended to four mutants of fumarate reductase, impaired by single amino acid substitutions at either of the putative quinone binding sites (QA or QB) of the enzyme. The results were analyzed in terms of the model of these dual sites of quinone binding in fumarate reductase, as well as the nature of the substituent in the 2-position of the dinitrophenol inhibitors.

Inhibition of NADH dehydrogenase (Complex I) of the mitochondrial respiratory chain by 1-methyl-4-phenylpyridinium (MPP+) and its analogs results in dopaminergic cell death. In the present study, the inhibition of mitochondrial respiration and of NADH oxidation in inverted inner membrane preparations by the oxidation products of N-methyl-stilbazoles (N-methyl-styrylpyridiniums) are characterized. These nonflexible MPP+ analogs were found to be considerably more potent inhibitors than the corresponding MPP+ derivatives. The IC50 values for these compounds and previously published figures for MPP+ analogs were then used to select a computer model based on structural parameters to predict the inhibitory potency of other compounds that react at the "rotenone site" in Complex I. A series of 12 novel inhibitors different in structure from the basic set were used to test the predictive capacity of the models selected. Despite major structural differences between the novel test compounds and the MPP+ and styrylpyridinium analogs on which the models were based, substantial agreement was found between the predicted and experimentally determined IC50 values. The value of this technique lies in the potential for the prediction of the inhibitory potency of other drugs and toxins which block mitochondrial respiration by interacting at the rotenone sites.

This paper examines the experimental foundations of reports in the literature on mitochondrial diseases involving Complexes I and II 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-1,2,3,6-tetrahydropyridine (MPTP), which produces biochemical, pharmacological, and clinical syndromes closely resembling Parkinsonism, the etiology of the disease is examined.

The two forms of monoamine oxidase (MAO), A and B, continue to be of major interest to biochemists, pharmacologists, neurologists, and gerontologists. Despite intensive study for more than half a century, unexpected and unique properties of these enzymes continue to come to light. Recent studies have centered on their kinetic mechanism, their unique predilection for substrates related to the neurotoxic tertiary amine MPTP, and their putative role in aging and in the etiology of neurodegenerative diseases. New and potent inhibitors of MAO A and MAO B continue to be developed because of their potential use in clinical medicine. Some are effective in the picomolar range but MAO B from different mammalian species shows remarkable differences in sensitivity to these agents.

Oxadiazolones and oxadiazolethiones are potent, reversible competitive inhibitors of MAO B in rat brain mitochondria. We have compared the K i values of six of these inhibitors toward MAO B from rat, human and beef liver mitochondria, using benzylamine as substrate. Unexpectedly, their inhibitory potency varies by 3 to 4 orders of magnitude between rat and beef liver MAO B, whereas the inhibition of the rat and human liver enzymes is quite similar. Examples are 5-(4-benzyl-oxyphenyl)-3-(2-cyano-ethyl)-1,3,4-oxadiazole-2(3 H )- thione, with K i at 30° of 0.5 nM for rat, 0.8 nM for human, and 1,830 nM for beef liver mitochondria and 5-(4-benzyloxyphenyl)-3-(2-hydroxyethyl)-1,3,4-oxadiazol-2(3 H )- one with K i values of 1.2, 1.1 and 1,320 nM for MAO B from these three sources. Since solubilized and membrane-bound enzymes had similar sensitivities to the inhibitors, the differences seen must arise from differences in the amino acid sequences of the three enzymes.

Flavin-containing monoamine oxidases (MAO A and MAO B) located on the outer membrane of mitochondria oxidise amines and generate hydrogen peroxide. Inhibitors alleviate depression by increasing neurotransmitter levels in the brain. Elevation of neurotransmitters,although an established outcome, is a delicate balance because complete lack of MAO A is associated with aggression and combination of monoamine oxidase inhibitors with reuptake inhibitors can result in serotonin toxicity. MAO in the periphery is essential for protection against biogenic amines, so inhibition there is an undesirable side effect both of antidepressants and drugs for other targets.MAO also metabolizes many amine drugs, an important factor in pharmacokinetics. This review summarises the structure, assay and regulation of MAO. The importance of reliable inhibition data properly analysed for these flavoenzymes is emphasised. It describes some current drugs and how new compounds that inhibit MAO are emerging from structure-based drug design.