Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders

Hao Lu; Lisa Kopcho; Kaushik Ghosh; Mark Witmer; Michael Parker; Sumit Gupta; Marilyn Paul; Prasad Krishnamurthy; Basanth Laksmaiah; Dianlin Xie; Jeffrey Tredup; Litao Zhang; Lynn M Abell

doi:10.1016/j.ab.2016.02.003

Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders

Anal Biochem. 2016 May 15:501:56-65. doi: 10.1016/j.ab.2016.02.003. Epub 2016 Feb 11.

Authors

Affiliations

¹ Lead Discovery and Optimization, Bristol-Myers Squibb R&D, Pennington, NJ 08534, USA. Electronic address: [email protected].
² Lead Discovery and Optimization, Bristol-Myers Squibb R&D, Pennington, NJ 08534, USA.
³ Disease Sciences and Technology, Biocon Bristol-Myers Squibb R&D Centre, Bangalore, 560099, India.
⁴ Protein Science, Bristol-Myers Squibb R&D, Princeton, NJ 08648, USA.
⁵ Discovery Chemistry, Bristol-Myers Squibb R&D, Wallingford, CT 06492, USA.
⁶ Lead Discovery and Optimization, Bristol-Myers Squibb R&D, Pennington, NJ 08534, USA. Electronic address: [email protected].

PMID: 26874021
DOI: 10.1016/j.ab.2016.02.003

Abstract

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.

Keywords: Fluorescence; High throughput; KATII; Mass spectrometry; RapidFire.

MeSH terms

Brain / drug effects
Brain / metabolism
Central Nervous System Diseases / drug therapy
Central Nervous System Diseases / enzymology
Drug Evaluation, Preclinical / methods*
Enzyme Assays / methods*
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
High-Throughput Screening Assays / methods
Humans
Kynurenic Acid / metabolism
Mass Spectrometry / methods
Spectrometry, Fluorescence / methods
Transaminases / antagonists & inhibitors*
Transaminases / metabolism

Substances

Enzyme Inhibitors
Transaminases
kynurenine-oxoglutarate transaminase
Kynurenic Acid