Advances in Bioanalytics and Biomarkers
Target Engagement and Pathways
Stable isotope labeled substrates can be of broad use in cases where target-based high-throughput screening aims to identify compounds that can modulate enzyme activity. For example, depending on the source of a given enzyme target, the presence of endogenous substrates or products can limit one’s ability to follow substrate®product conversions; utilization of a labeled substrate(s) can help overcome background contamination. These same isotope flux assays can then be used to follow the progression of hits in later stages of development, including cell-based assays and in vivo studies. Although stable isotope tracer kinetics, coupled with mass spectrometry-based detection, can therefore connect all phases of drug discovery there are caveats that should be recognized to ensure reliable data interpretations.
Our presentation will highlight key areas where the logic surrounding tracer kinetics diverges as the application of flux analyses moves across different stages of drug discovery. We will consider a case study that is focused on lipid biology, i.e. modulating the level of glycosylated ceramides. We will first outline how labeled substrates can be used to circumvent problems that arise in early screening. We will then outline how tracers can be used to progress molecules into later phases, including in vivo studies. Although one can use virtually the same back-end mass spectrometry assay to measure the formation of labeled products, several parameters change with regards to dosing the labeled substrates. For example, when measuring enzyme activity in early biochemical screening one needs to only measure the labeled product. In contrast, in vivo studies must contend with the fact that substantial amounts of “cold” (endogenous) substrate can exist, in addition, it may not be possible to maintain a steady-state exposure to the labeled substrate. Consequently, strategies need to account for temporal tracer dilution, most of which may not be immediately obvious and/or difficult to correct.
In summary, the ability to measure stable isotope flux from precursors to products can provide a bridge that spans the entire spectrum of drug discovery and development. However, changes in the generation of a labeled product do not immediately reflect changes in the metabolic activity of a given target enzyme or pathway, it is possible to observe differences in the abundance of a labeled product which reflect an unexpected modulation of precursor metabolism. Although the example described here is focused on a targeted screen, the logic has immediate implications with regards to phenotypic screening; attention to a few details can influence essential decision points.