Details

Autor(en) / Beteiligte

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

Titel

Lead discovery for human kynurenine 3-monooxygenase by high-throughput RapidFire mass spectrometry

Ist Teil von

• Journal of biomolecular screening, 2014-04, Vol.19 (4), p.508-515

Ort / Verlag

United States

Erscheinungsjahr

2014

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.