Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

Denise M. Lowe, Michelle Gee, Carl Haslam, Bill Leavens, Erica Christodoulou, Paul Hissey, Philip Hardwicke, Argyrides Argyrou, Scott P. Webster, Damian J. Mole, Kris Wilson, Margaret Binnie, Beverley A. Yard, Tony Dean, John Liddle, Iain Uings, Jonathan P. Hutchinson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z ' value 0.8) and provided several tractable hit series for further investigation.

Original languageEnglish
Pages (from-to)508-515
Number of pages8
JournalJournal of Biomolecular Screening
Volume19
Issue number4
DOIs
Publication statusPublished - Apr 2014

Keywords

  • high-throughput screening
  • RapidFire mass spectrometry
  • kynurenine 3-monooxygenase
  • kynurenine
  • 3-hydroxykynurenine
  • INHIBITORS
  • 3-HYDROXYLASE
  • EXPRESSION
  • PATHWAY
  • ASSAYS
  • BRAIN

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