Advances in Bioanalytics and Biomarkers
Target Engagement and Pathways
Metabolism and in particular central energy metabolism have evolved as promising drug targets. A cutting edge technology which has become widely used for studying oxygen consumption and extracellular acidification is the Seahorse™ analyzer. While this technology allows for rapid label free screening it does not provide further details on the involved metabolites and pathways. The quantitative analysis of these pathways, mainly involving glycolysis, the tricarboxylic acid cycle (TCA) and adjacent pathways is intrinsically very challenging. Several commercial solutions have evolved over the years predominantly using mass spectrometry and a series of labeled internal standards. However, many of these approaches suffer from long analytical procedures and the need for special internal standards or kits.
As an alternative we will discuss our NMR based workflow allowing the quantitative analysis of several important pathways as for example glycolysis, TCA cycle, OxPhos, one-carbon metabolism and others. Our NMR based workflow allows for the rapid and quantitative analysis of >80 metabolites without the need for specialized kits or internal standards. The workflow can partially be operated in an automated fashion using a KNIME workflow such as KIMBLE. Moreover, flux analysis using 13C labeled materials can easily be adapted resulting in kinetic information.
As an example for the usefulness of this workflow we will discuss the discovery of choline kinase α (CHKA) as a possible target for the prevention of epithelial to mesenchymal transition (EMT) an important metastatic process. Using NMR based analysis of metabolic changes during TGFβ induced EMT we could observe significant alterations in choline phosphorylation. Following up on these results by using experimental inhibitors we could identify CHKA as crucial enzyme for the EMT phenotype.