SLAS

Poster: A Novel High Throughput Drug Screening Technique Targeting Human Kynurenine 3-Monooxygenase

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Poster presentation at SLAS2014.
Winner of a SLAS Young Scientist Award.

Kris Wilson BSc (Hons)
University of Edinburgh

Poster Presenter SLAS2014 Kris Wilson.jpg
Following a BSc in Biology at the University of Stirling I took up the position of research scientist at Scottish Biomedical (a small drug discovery company based in Glasgow) where I worked on discovery of novel phosphodiesterase 10 (PDE10) inhibitors for the treatment of schizophrenia, amongst other projects. Following this, I joined the University of Edinburgh’s drug discovery team as a research technician working on novel drug targets for obesity and multi-organ failure. I have since undertaken a PhD project which is now close to completion. My PhD is a collaborative project based within the drug discovery, chemical and translational biology and inflammation research groups at the university.


Abstract

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the tryptophan degradative pathway. KMO is emerging as an increasingly important target for drug development.  The enzyme is implicated in the development and progression of several neurodegenerative disorders and in the regulation of the immune response.  Production of recombinant human enzyme is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems.  Although several in vitro KMO assay techniques have been reported in the literature these were developed using more readily expressed rat (Rattus norvegicus) and bacterial (Pseudomonas fluorescens) KMO enzyme.

This is the first report of successful bacterial expression of soluble active human KMO enzyme.  Cleavable fusion protein tags were used to optimise solubility and enable characterisation and partial purification of this active construct.  This source of functional enzyme was used to develop a novel high throughput drug screening technique for the discovery of inhibitors specifically targeting human KMO.  This screening technique is a magnetic bead based assay with LC-MS readout which identifies compounds with specific binding affinity for human KMO enzyme. The assay demonstrated a Z’ factor of 0.74 and was fully validated using known KMO inhibitors from the patent literature.  Importantly, this assay avoids the issues associated with handling and preparation of human KMO as it does not require purified protein.

Bacterial expression of the human enzyme represents progress towards the elucidation of the human KMO crystal structure and has enabled screening of potential new drugs directly against the human enzyme.  This will allow accelerated and improved discovery and development of novel KMO inhibitors for the benefit of numerous disease states.