Lead discovery for human kynurenine 3-monooxygenase by high-throughput RapidFire mass spectrometry
| Autor: | Bill Leavens, Jonathan P. Hutchinson, Carl Haslam, Damian J. Mole, John Liddle, Scott P. Webster, Beverley A. Yard, Argyrides Argyrou, Erica Christodoulou, Margaret Binnie, Paul Hissey, P. Hardwicke, Kris Wilson, Denise M. Lowe, Tony W. Dean, Iain Uings, Michelle Gee |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
High-throughput screening
3-Hydroxykynurenine Drug Evaluation Preclinical Biology Mass spectrometry Biochemistry Mass Spectrometry Analytical Chemistry Cell Line chemistry.chemical_compound Kynurenine 3-Monooxygenase Catalytic Domain Drug Discovery Animals Humans Enzyme Inhibitors Chromatography Tryptophan Substrate (chemistry) Recombinant Proteins High-Throughput Screening Assays Mass Enzyme Activation Kinetics chemistry Molecular Medicine Kynurenine Biotechnology Protein Binding |
| Zdroj: | Journal of biomolecular screening. 19(4) |
| ISSN: | 1552-454X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|