Demonstration of Isoleucine 199 as a Structural Determinant for the Selective Inhibition of Human Monoamine Oxidase B by Specific Reversible Inhibitors

Abstract

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO Two isoforms of monoamine oxidase (MAO),1 MAO A and MAO B, exist in humans and are both ϳ60-kDa outer-mitochondrial membrane-bound flavoenzymes that share ϳ70% sequence identities (1). Because these enzymes have distinct and overlapping specificities in the oxidative deamination of neurotransmitters and dietary amines, the development of specific reversible inhibitors has been a long sought goal. Expression levels of MAO B in neuronal tissue increase ϳ4-fold with age (2), resulting in an increased level of dopamine metabolism and the production of higher levels of hydrogen peroxide, which are thought to play a role in the etiology of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases (3). Thus, the development of specific, reversible MAO B inhibitors could lead to clinically useful neuroprotective agents.Recent studies in the literature have shown that 8-(3-chlorostyryl)caffeine (CSC), an A 2A adenosine receptor antagonist, is also a potent and selective inhibitor of mouse brain MAO B (K i ϭ 100 nM) but not MAO A (4). In addition, trans,trans-farnesol, a component of tobacco smoke, is a potent, reversible inhibitor specific for MAO B.2 Another study has established that 1,4-diphenyl-2-butene (K i ϭ 35 M), a contaminant of polystyrene bridges used for MAO B crystallization, and 1,4-diphenyl-1,3-butadiene (K i ϭ 7 M) are potent, competitive MAO B-specific reversible inhibitors (6, 7). Because none of these compounds (see Scheme 1 for their respective structures) inhibit MAO A, a more detailed study of the molecular basis for their specificities could provide opportunities to develop MAO B-selective inhibitors with potential neuroprotective properties.Recent crystal structures of human MAO B in complex with several pharmacologically important inhibitors have been solved to 1.6-Å resolution (6,8). The access channel from the surface of the protein to the active site of the enzyme consists of two cavities, the entrance cavity and the active site cavity (9). Depending on the nature of the inhibitory species, these cavities are either separate or fused depending on the conformation of the Ile-199 "gate" residue. The analogous position in MAO A is known from sequence comparisons to be Phe-208. The recently reported structure of MAO A (10) is at a resolution (3.2 Å) that precludes a detailed molecular description of the properties of the active site that would allow a comparison with that of human MAO B. Previous studies on the catalytic and inhibitor properties of rat (11) and of human (12) I199F MAO B mutant proteins show clear differences with the respective native enzymes which could not be readily interpreted in molecular detail. * This work was supported by Grant GM-29433 from NIGMS, National Institutes of Health, by Ministry of Science and Education (Italy) Grants FIRB and COFIN04, and by Fondazione MINTAS. The costs of publication of this article were defrayed in part by the payment of page c...

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

“…However the value of the kinetic parameter, k +2 , for covalent adduct formation with ox liver MAO- Thus pheniprazine is a significantly better mechanismbased inhibitor of MAO-B from ox liver than of either form of the enzyme from rat liver. Such results are consistent with those of others indicating that there are species differences in the substrate specificities and inhibitor sensitivities of MAO-B [16][17][18][19][20]. The similar binding affinities of rat liver MAO-A and ox liver MAO-B may result from the specific change of the residue at position 199 that occurs in the ox enzyme.…”

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

“…The recombinant rMAOB obtained from P. pastoris expression system exhibits similar K m value for benzylamine (Table 3) as reported earlier for the enzyme purified from S. cerevisiae expression system [9]. The purification scheme discussed in Methods section is modified for pH and buffer conditions relative to that previously published for human MAOA and MAOB [13,14,19]. As mentioned above, unlike human MAOA and MAOB, purification of rat MAOB requires using a higher pH (7.5-7.6) for efficient binding and subsequent washings in the anion exchange high-Q column.…”

“…Purification of rMAOB was done following the hMAOB protocol [13,19] with some minor modifications to improve the yield and purity of the enzyme. Approximately 130 gram (wet cell weight) of P. pastoris cells (ca.…”

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

“…Farnesylamine was used as a substrate because previous structural data show bound farnesol to traverse both entrance and substrate cavities (23) so that any inhibition observed by 2-BFI would occur by its binding to only the free form of the enzyme. (A more comprehensive kinetic analysis of 2-BFI inhibition will be reported elsewhere.)…”