Impact of Cell Background on Small Compound Target Deconvolution by CETSA Coupled to Mass Spectrometry

The cellular thermal shift assay (CETSA) is a powerful tool for confirming target engagement and deconvoluting mode of action in living cells. The principle behind CETSA is that a ligand – target protein interaction changes the thermal stability of the ligated protein and this can be assessed by comparing ligand treated cells to nontreated cells after a heat challenge. By employing mass spectrometry (MS) detection after CETSA (CETSA®Explore/PISA) we have been able to extend the changes in thermal stability to more proteins than just the direct targets, also those proteins that are part of the same metabolic/signaling pathways as the target can display changes in thermal stability. If the effects on the cell are profound, also protein stability changes associated with a change in phenotype can be detected, for example changes in intracellular vesicle dynamics.

In an effort to better understand how drugs perturb living cells we developed the target engagement atlas (TEA). In this CETSA based screening effort we profiled over 400 small molecular weight compounds , comprising old and recently approved drugs, obsolete/withdrawn drugs, and natural products, as well as probes described to have a certain function. With the needs of the pharmaceutical industry in mind we profiled all of the compounds in HepG2 cells, but a subset of compounds was also investigated in other cell lines. For example, we profiled kinase inhibitor responses in different cell backgrounds and performed a focused study on five breast cancer cell lines and assessed their CETSA responses to eight currently used therapeutic compounds. In the work presented here we will show how the TMT based MS workflow has enabled our studies of how different compounds exert cell background specific responses.