Hydrophobic Interaction Liquid Chromatography Mass Spectrometry Enables Cellular Assay Development to Identify Inhibitors of Glycine N-Methyltransferase

Authors

• JH Shrimp
• QM Hanson
• N Hoxie
• J Janiszewski
• V Chenniappan
• C Kelly
• M Shen
• S Patnaik
• MD Hall

Abstract

A factor impacting clinical end points is robust on-target activity from a drug. Therefore, it is important to demonstrate on-target activity of drug candidates within cells during preclinical development and to not only rely on activity from biophysical and biochemical assays using isolated purified protein. Mass spectrometry can be used to demonstrate on-target activity within cells. Glycine N-methyltransferase (GNMT) catalyzes the methylation of glycine to sarcosine using S-adenosyl-L-methionine (SAM) as a cofactor. We spiked D2-labeled glycine into cell culture media to quantitively monitor its conversion D2-sarcosine (92/45 m/z) by GNMT. GNMT is a critical liver enzyme that regulates SAM levels. We set out to develop mass spectrometry-based methods capable of profiling GNMT inhibition in 384-well biochemical and cellular assays.
The cellular assay consisted of HEK293T cells overexpressing GNMT. To distinguish the GNMT reaction product sarcosine from the endogenous isomeric metabolites (alanine and sarcosine), the assay modified DMEM to contain 0.4 mM D2-glycine (as the only media source of glycine). The D2-glycine is converted to D2-sarcosine within the cell, and the quantity of D2-sarcosine converted reports on the activity of GNMT. A 2X30mm BEH-Amide column was used to retain sarcosine following reaction quench with acetonitrile containing 0.5 µM, 13C315N-labeled sarcosine as internal standard. A 1-min linear gradient was established on a model 1290 UHPLC pump (Agilent), coupled with LS1 autosampler and Sciex 6500 tandem mass spectrometer to enable lead generation screening. The overall cycle time was 2-min per sample.

Scientific Focus Area: Molecular Biology and Biochemistry

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