Development of LC/MS/MS, High-Throughput Enzymatic and Cellular Assays for the Characterization of Compounds That Inhibit Kynurenine Monooxygenase (KMO)

Abstract

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.

Lead Discovery for Human Kynurenine 3-Monooxygenase by High-Throughput RapidFire Mass Spectrometry

“…The K m value for Kyn is comparable to reported values of 13 µM for a crude extract of recombinant human KMO expressed in mammalian cells 21 and 14 µM for KMO obtained from human liver extracts. 13 Considerably higher K m values have been reported for both substrates against human KMO which had been expressed in mammalian cells and solubilized and purified in the presence of 0.2% Triton X-100. 10 Treatment of the Sf9-derived KMO with a membrane-solubilizing concentration of detergent (0.01% n-dodecyl-β-D-maltoside) also resulted in an elevation of the K m values for both substrates, to 42 µM for Kyn and 2 µM for NADPH, suggesting that the membrane environment is involved in substrate binding.…”

“…19 In the recently published work of Winkler et al, 13 KMO activity was obtained by subcellular fractionation of liver tissue from various species, and relatively high concentrations of the resulting material were required for assay (~0.1 mg/mL total protein). To provide a consistent supply of KMO activity for an extended hit to lead campaign and avoid the need to obtain human tissue, recombinant expression was assessed.…”

“…Recently, the use of liquid chromatographytandem mass spectrometry was reported as a sensitive, label-free, and direct method for monitoring the conversion of Kyn to 3-HK, which was successfully applied to the assay of KMO activity in tissue extracts as well as cellular assays with a throughput of approximately 90 s per sample. 13 We describe here a complementary application of mass spectrometry to the measurement of KMO activity, of approximately 10-fold higher throughput, using the RapidFire high-throughput mass spectrometry (RF-MS) system (Agilent Technologies, Lexington, MA). This enabling technology has previously been successfully used in lead discovery campaigns for challenging targets.…”

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease

“…Selectivity of CHDI-340246 against KAT isoforms, kynureninase and IDO have been previously reported (referred to as compound 75 in (Toledo-Sherman et al, 2015;Winkler et al, 2013). In the present study, additional radioligand displacement assays with 10 µM CHDI-340246 were conducted against a diverse panel of CNS and other peripheral targets was performed to assess off-target liability (Cerep, France).…”

“…Secondly, that the elevation of Kyn and KynA, on their own, would confer some benefit in HD models due to their neuroactive properties. We therefore developed an orally-bioavailable, potent and selective KMO inhibitor, CHDI-340246, to rigorously evaluate the involvement of KMO in disease progression (Toledo-Sherman et al, 2015;Winkler et al, 2013). We show a dose-dependent modulation of the KP metabolites in blood and in multiple tissues, including brain, after oral administration and provide a detailed quantitative pharmacokinetic-pharmacodynamic characterization of drug activity.…”