International Journal of Molecular Sciences Article Hydride Abstraction as the Rate-Limiting Step of the Irreversible Inhibition of Monoamine Oxidase B by Rasagiline and Selegiline: A Computational Empirical Valence Bond Study Tana Tandari´c 1, Alja Prah 2,3, Jernej Stare 2, Janez Mavri 2 and Robert Vianello 1,* 1 Division of Organic Chemistry and Biochemistry, Ruder¯ Boškovi´cInstitute, BijeniˇckaCesta 54, HR-10000 Zagreb, Croatia; [email protected] 2 Laboratory for Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia; [email protected] (A.P.); [email protected] (J.S.); [email protected] (J.M.) 3 Faculty of Pharmacy, University of Ljubljana, AškerˇcevaCesta 7, SI-1000 Ljubljana, Slovenia * Correspondence: [email protected] Received: 21 July 2020; Accepted: 21 August 2020; Published: 26 August 2020 Abstract: Monoamine oxidases (MAOs) catalyze the degradation of a very broad range of biogenic and dietary amines including many neurotransmitters in the brain, whose imbalance is extensively linked with the biochemical pathology of various neurological disorders, and are, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. Still, despite this practical significance, the precise molecular mechanism underlying the irreversible MAO inhibition with clinically used propargylamine inhibitors rasagiline and selegiline is still not unambiguously determined, which hinders the rational design of improved inhibitors devoid of side effects current drugs are experiencing. To address this challenge, we present empirical valence bond QM/MM simulations of the rate-limiting step of the MAO inhibition involving the hydride anion transfer from the inhibitor α-carbon onto the N5 atom of the flavin adenin dinucleotide (FAD) cofactor. The proposed mechanism is strongly supported by the obtained free energy profiles, which confirm a higher reactivity of selegiline over rasagiline, while the calculated difference in the activation Gibbs 1 energies of DDGz = 3.1 kcal mol− is found to be in very good agreement with that from the measured 1 literature kinact values that predict a 1.7 kcal mol− higher selegiline reactivity. Given the similarity with the hydride transfer mechanism during the MAO catalytic activity, these results verify that both rasagiline and selegiline are mechanism-based irreversible inhibitors and offer guidelines in designing new and improved inhibitors, which are all clinically employed in treating a variety of neuropsychiatric and neurodegenerative conditions. Keywords: irreversible inhibition; monoamine oxidase; hydride transfer; antiparkinsonian drugs; neurodegeneration; flavoenzymes 1. Introduction Monoamine oxidases (MAOs) are flavoenzymes that metabolize a wide range of biogenic and dietary amines, including monoamine neurotransmitters, such as dopamine, serotonin, and adrenaline, in both the central nervous system and peripheral tissues [1]. Proper degradation of these molecules is responsible for the stable function of synaptic neurotransmission, resulting in a healthy brain condition. Since different concentrations of these small monoaminergic neurotransmitters influence our mood and emotions, as well as the control of motor, perceptual, and cognitive functions, any imbalance in their Int. J. Mol. Sci. 2020, 21, 6151; doi:10.3390/ijms21176151 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 6151 2 of 13 concentration leads to the development and progression of a number of, if not all, neuropsychiatric andInt. J. neurological Mol. Sci. 2020, 21 diseases., x FOR PEER REVIEW 2 of 13 Int. J. ThereMol. Sci. are 2020 two, 21, x known FOR PEER isoforms REVIEW of the MAO enzyme—MAO A and MAO B [2,3]—which share2 of 13 There are two known isoforms of the MAO enzyme—MAO A and MAO B [2,3]—which share around 70% sequence identity and the same FAD cofactor covalently attached at a conserved cysteine around 70% sequence identity and the same FAD cofactor covalently attached at a conserved cysteine residueThere [4]. Theirare two active known site cavitiesisoforms are of placed the MAO deep enzyme—MAO inside the enzyme, A and and MAO differ B in [2,3]—which volume and shapeshare residue [4]. Their active site cavities are placed deep inside the enzyme, and differ in volume and betweenaround 70% isoforms sequence [5], identity which results and the in same different FADsubstrate cofactor covalently and inhibitor attached affinities at a conserved [6]. The chemical cysteine shape between isoforms [5], which results in different substrate and inhibitor affinities [6]. The reactionresidue [4]. catalyzed Their active by both site MAO cavities isoforms are placed involves deep the inside oxidation the enzyme, of the amine and differ moiety in via volume oxidative and chemical reaction catalyzed by both MAO isoforms involves the oxidation of the amine moiety via cleavageshape between of the substrate isoformsα [5],-CH which bond whichresults consequently in different generatessubstrate anand imine inhibitor intermediate. affinities This [6]. stepThe chemicaloxidative reaction cleavage catalyzed of the substrate by both α -CHMAO bond isoforms which involves consequently the oxidation generates of an the imine amine intermediate. moiety via is accomplished by the reduction of FAD to FADH2 that is followed by reoxidation back to FAD oxidativeThis step cleavageis accomplished of the substrate by the reduction α-CH bond of FAD which to consequentlyFADH2 that is ge followednerates anby imine reoxidation intermediate. back to with molecular oxygen and a simultaneous H2O2 release. The imine intermediate is hydrolyzed FAD with molecular oxygen and a simultaneous H2O2 release. The imine intermediate is hydrolyzed nonenzymaticallyThis step is accomplished to the corresponding by the reduction aldehyde of FAD and to ammonia FADH2 that (with is primaryfollowed amines) by reoxidation or a substituted back to nonenzymatically to the corresponding aldehyde and ammonia (with primary amines) or a amineFAD with (from molecular secondary oxygen amines) and [7 a]. simultaneous The overall catalytic H2O2 release. MAO The reaction imine is intermediate given as: is hydrolyzed nonenzymaticallysubstituted amine (fromto the secondary corresponding amines) aldehyde [7]. The overalland ammonia catalytic MAO(with reaction primary is givenamines) as: or a substituted amine (from secondary amines) [7]. The overall catalytic MAO reaction is given as: All products of the MAO-mediated reactions are highly reactive species, and include a number of potentiallyAllAll productsproducts neurotoxic ofof the the MAO-mediated MAO-mediated agents [8–10]. reactionsBecause reactions of are arethat, highly highly constant reactive reactive excessive species, species, activity and and include includeof MAO a number a enzymes number of potentiallyofmay potentially lead to neurotoxic mitochondrialneurotoxic agents agents damages [8 –[8–10].10]. 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