Development of Spin-Labeled Pargyline Analogues as Specific Inhibitors of Human Monoamine Oxidases A and B

Abstract

Three TEMPO conjugated pargyline analogues (ParSL-1, ParSL-2 and ParSL-3) have been synthesized and their inhibitory properties tested for the two human monoamine oxidase isoforms (hMAOA and hMAOB). The three analogues differ in flexibility and substituent positions (para or meta) of the linkers connecting the TEMPO group to the pargyline phenyl ring. ParSL-1 contains a flexible acetamido (-CH 2 -CO-NH-) linker connecting the two moieties at the para position. In contrast, the TEMPO moieties in ParSL-2 and ParSL-3 are attached with rigid amido (-CO-NH-) linkers to the para or meta positions of the pargyline phenyl ring, respectively. These variations in conformational flexibility and substituent position are shown to have profound effects in tuning the specificities of these analogues towards the two MAO isoforms. ParSL-1 irreversibly inhibits either MAOA and MAOB, ParSL-2 inhibits only MAOB (K i = 15 ± 5 μM), and ParSL-3 is found to be specific for MAOA (K i = 268 ± 72 μM). These results thus provide additional insights into the role of conformational flexibility and structural properties of MAO inhibitors in tuning their isoform specificities. These active site probes have been used to determine the topological orientation of these enzymes in the mitochondrial membrane. Studies with intact mitochondria show MAOA is topologically on the cytosolic face of the outer membrane in human placenta but recombinant MAOA is situated on the opposite inner face in Pichia mitochondria. Recombinant MAOB is found to be situated on the cytosolic face of the outer membrane in Pichia mitochondria.Of the spectroscopic probes available for membrane bound proteins, nitroxide spin labels are considered the most useful in defining structural properties of these important class of proteins. With advances in EPR and NMR methodologies, the design of enzyme-specific paramagnetic spin probes will allow understanding structural properties of these proteins in their native membrane-bound forms which can be extended to tissues samples as well as to subcellular organelles. Recent studies on human and rat monoamine oxidases (MAOA and MAOB) utilized a MAO -specific pargyline analogue containing a TEMPO spin label to show differences in active site accessibility (1) as well as demonstrated both isoforms are dimeric in their respective membrane bound forms in the outer mitochondrial membrane (OMM) (2). The crystal structures of hMAOA (3,4) and hMAOB (5) exhibit overall similarities in Cα-protein folds, as expected from their high sequence identities (ca. 70%). However, the structural properties of their active site cavities are quite different. The active site of hMAOB is dipartite with a substrate binding cavity (420 Å 3 ) and a substrate entrance cavity (290 Å 3 ) (5) separated by Ile199, whose side chain functions as a gate in regulating substrate/inhibitor binding (6). In contrast, the active site in hMAOA consists of a single substrate binding cavity (550 Å 3 ), which appears to provide more conformational flexibility for bound ligan...

Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis

“…As shown in Table 1, these pargyline analogues bind to rat MAO A with K i values ranging from 65–165 μM and to rat MAO B with K i values ranging from 32–250 μM. ParSL-1 and ParSL-2 inhibit human MAO B with K i values of 22 and 15 μM, respectively with only ParSL-1 demonstrating binding to human MAO A (K i =212 μM) (14). ParSL-3 weakly binds to human MAO A (K i =268 μM) and exhibits no observable inhibition with human MAO B (14).…”

“…ParSL-1 and ParSL-2 inhibit human MAO B with K i values of 22 and 15 μM, respectively with only ParSL-1 demonstrating binding to human MAO A (K i =212 μM) (14). ParSL-3 weakly binds to human MAO A (K i =268 μM) and exhibits no observable inhibition with human MAO B (14). The data demonstrate differences in behavior of the rat enzymes as compared with the human enzymes on interaction with these pargyline analogues.…”

“…Percoll was obtained from Amersham Biosciences. The TEMPO-substituted pargyline analogues (ParSL-1, ParSL-2, and ParSL-3) were synthesized by Dr. Anup Upadhyay in this laboratory as described previously (14). All other chemical used in this work were purchased from Sigma-Aldrich.…”

“…Previous published work from this laboratory (14) has shown that TEMPO-substituted pargyline (ParSL1–3, structures in Figure S1, Supplementary Materials) analogues exhibit differential reactivities with human MAO A and MAO B depending on whether the TEMPO moiety is in the meta (ParSL-3) or para (ParSL-2) amide linkages with the benzene ring whereas ParSL-1 inactivates either enzyme (14). It was also demonstrated that, in intact mitochondria isolated from the expression strain of Pichia pastoris , human MAO B is readily inactivated by a ParSL-1 whereas human MAO A is unreactive.…”

Synthesis of some novel 2-substituted benzothiazole derivatives containing benzylamine moiety as monoamine oxidase inhibitory agents

“…Hence, it may conceivably be declared that arylalkylamine or benzylamine moieties contest with endogenic amines. This consideration has been supported by some studies indicating that benzylamine moiety possesses MAO inhibitory activity [20][21][22] . In addition to benzylamine, the compounds containing benzothiazole moiety have been reported to inhibit MAO enzymes 23 .…”