Discussion

Understanding drug mechanism poses the daunting challenge of determining the affinity of the drug for all potential targets. Drug target engagement can be assessed by a cellular thermal shift assay (CETSA) based on ligand-induced changes in protein thermal stability (1). We combined the CETSA method with quantitative mass spectrometry to study the effect of drugs on the thermal profile of a cellular proteome comprising over 7000 proteins (2). The approach, termed proteome profiling (TPP), enabled the unbiased monitoring of drug targets, off-targets, and downstream effectors. To rank binding affinities among multiple targets, we determined stability profiles across a range of compound concentrations at a defined temperature. Comparison of the thermal profiles obtained after drug treatment of intact cells versus cell extract allowed us to distinguish effects induced by ligand binding from those induced by downstream modifications.

(1) Martinez-Molina et al., Science, 2013. doi: 10.1126/science.1233606

(2) Savitski et al., Science, 2014. doi: 10.1126/science.1255784