Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO)

J Biomol Screen. 2013 Sep;18(8):879-89. doi: 10.1177/1087057113489731. Epub 2013 May 20.


Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

Keywords: 3-hydroxykynurenine; LC/MS/MS; human PBMCs; kynurenine; kynurenine 3-monooxygenase; microglia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Line
  • Chromatography, Liquid / methods
  • Cricetulus
  • Dogs
  • Drug Discovery
  • Enzyme Assays / methods*
  • HEK293 Cells
  • Humans
  • Huntington Disease / drug therapy
  • Huntington Disease / metabolism
  • Kynurenine / analogs & derivatives
  • Kynurenine / biosynthesis
  • Kynurenine / metabolism
  • Kynurenine 3-Monooxygenase / antagonists & inhibitors*
  • Kynurenine 3-Monooxygenase / chemistry
  • Kynurenine 3-Monooxygenase / metabolism*
  • Leukocytes, Mononuclear / drug effects
  • Macaca fascicularis
  • Mice
  • Mice, Inbred C57BL
  • Rats
  • Rats, Wistar
  • Tandem Mass Spectrometry / methods


  • 3-hydroxykynurenine
  • Kynurenine
  • Kynurenine 3-Monooxygenase