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 means of a cellular thermal shift assay (CETSA) based on ligand-induced changes in protein thermal stability. We combined the CETSA method with quantitative mass spectrometry to study the effect of drugs on the thermal profile of a cellular proteome comprising more than 7000 proteins. The approach enabled the monitoring of drug targets and downstream effectors.

Tracking drugs in living cells. Drugs alter the thermal stability of proteins directly through compound binding or indirectly through changes in overall protein state. Thermal proteome profiling determines melting curves for thousands of proteins and tracks drug action in cells.

We devised a method for the thermal profiling of cellular proteomes. Cells were cultured with or without drugs and heated to different temperatures so as to induce protein denaturation, and remaining soluble proteins were extracted with buffer. At each temperature, soluble proteins were quantified by means of high-resolution mass spectrometry, yielding denaturation curves. This allowed determination of thermal stability and the identification of ligand-induced shifts. 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.

We performed thermal proteome profiling (TPP) on human K562 cells by heating intact cells or cell extracts and observed marked differences in melting properties between the two settings, with a trend toward increased protein stability in cell extract. Adenosine triphosphatase (ATP)–binding proteins showed a significant trend toward increased stability in intact cells, suggesting stabilization by the endogenous ligand. This was confirmed with the addition of ATP to cell extract, which resulted in increased stability for this protein group. The ability of TPP to identify target binding was validated by using the broad-specificity inhibitors staurosporine and GSK3182571, which induced shifts in the melting temperatures of many kinase targets and also affected the thermal profiles of other proteins, including regulatory subunits of kinase complexes. We identified the heme biosynthesis enzyme ferrochelatase (FECH) as an off-target of several kinase inhibitors and showed that the drug vemurafenib reaches full target occupancy of its cognate target BRAF and the off-target FECH within a narrow concentration window. FECH deficiency is genetically linked to protoporphyria, suggesting that the photosensitivity induced by vemurafenib and other drugs is mediated by FECH. Drug treatment of live cells affected not only direct target proteins but also downstream effectors. The ABL inhibitor dasatinib induced thermal shifts in several proteins downstream of BCR-ABL, including CRKL, and at concentrations in good agreement with the effect on cell growth.

Thermal profiling of cellular proteomes enables the differential assessment of protein ligand binding and other protein modifications, providing an unbiased measure of drug-target occupancy for multiple targets and facilitating the identification of markers for drug efficacy and toxicity.