title: Characterization of the binding of [3H]Ro 41-1049 to the active site of human monoamine oxidase-A.
abstract: The novel reversible and selective inhibitor of monoamine oxidase-A (MAO-A) Ro 41-1049 [N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide HCl] shows inhibition characteristics similar to those of the structurally related reversible MAO-B inhibitors Ro 16-6491 and Ro 19-6327. In the present study, tritiated Ro 41-1049 was used as a high affinity ligand to study the binding characteristics of this inhibitor to MAO-A and its interactions with the enzyme. An homogeneous population of high affinity binding sites for [3H]Ro 41-1049 was found in membrane preparations from human frontal cortex and placenta (Kd = 16.5 +/- 1.4 and 64.4 +/- 19.2 nM, respectively). In frontal cortex the Bmax value for [3H]Ro 41-1049 (2.6 +/- 0.4 pmol/mg of protein) was about one third of the Bmax calculated for the MAO-B-selective ligand [3H]Ro 16-6491. The density of [3H]Ro 41-1049 binding sites in human placenta varied greatly in the different tissue samples investigated, showing an average Bmax of 101.7 +/- 36.5 pmol/mg of protein. Apparent binding equilibrium was reached after 1 hr of incubation at 37 degrees. At this temperature the binding was reversible, with a dissociation t 1/2 of about 35 min. At lower temperatures the radioactivity dissociation was much slower. Among the various drugs tested, only inhibitors of MAO-A were able to effectively prevent [3H]Ro 41-1049 specific binding. As previously reported for the MAO-B ligands [3H]Ro 16-6491 and [3H]Ro 19-6327, the analysis of the membrane-bound radioactivity showed that [3H]Ro 41-1049 was entirely recovered in the form of its aldehyde derivative, indicating that Ro 41-1049 was deaminated by MAO-A. The existence of a Ro 41-1049 adduct reversibly bound to the enzyme active site might explain the inhibition mechanism of this compound. The exposure of the radioligand-enzyme complex to NaBH3CN at pH 4.5 caused the irreversible covalent incorporation of about 70% of the specifically bound radioactivity into a 60-kDa polypeptide. This incorporation was dependent on the pH and on the amount of NaBH3CN added. The presence of MAO-A- but not MAO-B-selective inhibitors prevented the covalent incorporation of [3H]Ro 41-1049. The present results indicate that [3H]Ro 41-1049 is incorporated into a subunit of MAO-A, in the presence of NaBH3CN, and modifies a protein domain that is essential for the enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of Milan, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Cesura
authors.forename: M
authors.initials: M
authors.lastname: Bös
authors.forename: M D
authors.initials: MD
authors.lastname: Galva
authors.forename: R
authors.initials: R
authors.lastname: Imhof
authors.forename: M
authors.initials: M
authors.lastname: Da Prada
mesh_terms: D000345:Affinity Labels; D001665:Binding Sites; D001667:Binding, Competitive; D002540:Cerebral Cortex / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D013329:Structure-Activity Relationship; D013844:Thiazoles / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D008996:Monoamine Oxidase Inhibitors; D013844:Thiazoles; C063456:Ro 41-1049; D008995:Monoamine Oxidase; D014357:Trypsin