title: Lead discovery for human kynurenine 3-monooxygenase by high-throughput RapidFire mass spectrometry.
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.
authors.affiliation: 1Department of Biological Sciences, GlaxoSmithKline, Stevenage, UK.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Lowe
authors.forename: Michelle
authors.initials: M
authors.lastname: Gee
authors.forename: Carl
authors.initials: C
authors.lastname: Haslam
authors.forename: Bill
authors.initials: B
authors.lastname: Leavens
authors.forename: Erica
authors.initials: E
authors.lastname: Christodoulou
authors.forename: Paul
authors.initials: P
authors.lastname: Hissey
authors.forename: Philip
authors.initials: P
authors.lastname: Hardwicke
authors.forename: Argyrides
authors.initials: A
authors.lastname: Argyrou
authors.forename: Scott P
authors.initials: SP
authors.lastname: Webster
authors.forename: Damian J
authors.initials: DJ
authors.lastname: Mole
authors.forename: Kris
authors.initials: K
authors.lastname: Wilson
authors.forename: Margaret
authors.initials: M
authors.lastname: Binnie
authors.forename: Beverley A
authors.initials: BA
authors.lastname: Yard
authors.forename: Tony
authors.initials: T
authors.lastname: Dean
authors.forename: John
authors.initials: J
authors.lastname: Liddle
authors.forename: Iain
authors.initials: I
authors.lastname: Uings
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Hutchinson
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D050601:Kynurenine 3-Monooxygenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D050601:Kynurenine 3-Monooxygenase
keywords: 3-hydroxykynurenine; RapidFire mass spectrometry; high-throughput screening; kynurenine; kynurenine 3-monooxygenase