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.

A Magnetic Bead–Based Ligand Binding Assay to Facilitate Human Kynurenine 3-Monooxygenase Drug Discovery

“…The inhibitory activities of these compounds against human KMO were verified in a separate kinetic assay. 5 The direct binding affinities of each compound for human KMO were generated using microdialysis, as described in the Microdialysis section. Compound 1 was assayed in the presence and absence of enzyme to generate the Z' factor for the magnetic bead-based assay.…”

“…Quantification of the product, 3-HK, by liquid chromatographymass spectrometry (LC-MS) analysis has been commonly used as an assay to establish the enzymatic activity of KMO. 5 This method has been demonstrated both in isolated protein extracts and in a cell-based context using KMO from several species. Oxidation of NADPH measured by spectrophotometry has also been applied to measure KMO activity for rat (Rattus norvegicus) and bacterial (Pseudomonas fluorescens) enzymes.…”

“…3 Therefore, there has been renewed interest in developing tractable in vitro assays for the identification of selective inhibitors. 5,6 Recombinant protein production of human KMO is problematic, with a number of solubility and purification difficulties reported in the literature. A large membrane targeting domain at the C-terminus of the human enzyme (amino acids 385-486) is understood to account for these difficulties.…”

Design and Implementation of High-Throughput Screening Assays

“…The mass spectrometry (MS) screening field in particular has made significant advances in recent years with the introduction of technologies such as RapidFire ® (Agilent) which couples a high-throughput autosampler/desalting step with an integrated ms/MALDI. This technology can now be used for medium-throughput screens with an average read time of <10 s/sample [14]. Additional technology development in this space is ongoing, and there are realistic prospects that throughput will be sufficient for full-deck HTS in the near future.…”

“…4 (a) Enzyme dilution (in μg protein/ml) curve for kynurenine monooxygenase (KMO). Enzyme activity was measured by mass spectrometry following the methodology described [14]. Open circle-0 μg/ml, filled diamond-4 μg/ml, open square-8 μg/ml, filled square-16 μg/ml, open triangle-32 μg/ml.…”