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

Abstract

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO.

Detecting Drug-Target Binding in Cells using Fluorescence Activated Cell Sorting Coupled with Mass Spectrometry Analysis

“…Compound 4 was confirmed to be inactive in the activity assay ( Figure 1). Cpds 1 -4 alongside the IC 50 values generated by us previously in a KMO activity assay against human enzyme [17] .…”

“…http://dx.doi.org/10.1101/121988 doi: bioRxiv preprint first posted online Apr. 3, 2017; This method was previously described by us [17] . The chromatographic system used was a TurboFlow TM TLX Aria-1 system (Thermo Fisher Scientific, Hemel Hempstead, UK), consisting of two Allegros pumps defined as the loading and eluting pumps, two valveswitching modules and a CTC liquid autosampler.…”

“…Cells were selected for two weeks using Hygromycin B (Sigma Aldrich) at 100 µg/mL to select for the hygromycin resistance gene contained within the pcDNA5 vector before positive colonies were isolated and cultured. HEK-huKMO cell lysate was analysed for KMO enzymatic activity by measuring the amount of KYN converted to 3HK detected using liquid chromatography-mass spectrometry (LC-MS/MS) following incubation with the compounds screened in the TAPS assay using a method we described previously [17] . Compounds were tested in a one compound per well format in this assay.…”

Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase

“…Wilson, et al 17 recently used kynurenine 3-monooxygenase immobilized on magnetic microbeads for MS-based screening of a commercial combinatorial library. Instead of exploiting the selectivity of mass spectrometry to screen combinatorial library mixtures or natural product extracts as in our approach, this group screened discreet compounds in series.…”

“…Instead of exploiting the selectivity of mass spectrometry to screen combinatorial library mixtures or natural product extracts as in our approach, this group screened discreet compounds in series. Also, instead of covalently attaching the enzyme as in our assays, Wilson, et al 17 covalently attached an antibody to the magnetic beads first and then used it to capture kynurenine 3-monooxygenase.…”