title: A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.
abstract: Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO.
authors.affiliation: Drug Discovery Core, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK kris.wilson@ed.ac.uk.
authors.forename: Kris
authors.initials: K
authors.lastname: Wilson
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.
authors.forename: Damian J
authors.initials: DJ
authors.lastname: Mole
authors.affiliation: Mass Spectrometry Core, Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.
authors.forename: Natalie Z M
authors.initials: NZ
authors.lastname: Homer
authors.affiliation: MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.
authors.forename: John P
authors.initials: JP
authors.lastname: Iredale
authors.affiliation: School of Biological Sciences and School of Biomedical Sciences, University of Edinburgh, C H Waddington Building, The University of Edinburgh, Edinburgh, UK.
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
authors.affiliation: Drug Discovery Core, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Webster
mesh_terms: D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D050601:Kynurenine 3-Monooxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D054852:Small Molecule Libraries
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D054852:Small Molecule Libraries; D050601:Kynurenine 3-Monooxygenase
keywords: drug discovery; inhibitors; kynurenine 3-monooxygenase; magnetic beads