A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery

Kris Wilson*, Damian J Mole, Natalie Z M Homer, John P Iredale, Manfred Auer, Scott P Webster

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)292-298
Number of pages7
JournalJournal of Biomolecular Screening
Issue number2
Early online date8 Oct 2014
Publication statusPublished - Feb 2015


  • drug discovery
  • inhibitors
  • kynurenine 3-monooxygenase
  • magnetic beads


Dive into the research topics of 'A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery'. Together they form a unique fingerprint.

Cite this