Research Article

Tracking cancer drugs in living cells by thermal profiling of the proteome

Science  03 Oct 2014:
Vol. 346, Issue 6205,
DOI: 10.1126/science.1255784

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Structured Abstract

INTRODUCTION

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.

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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.

RATIONALE

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.

RESULTS

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.

CONCLUSION

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.

Mapping human drug targets in the cell

To understand both the beneficial and the side effects of a drug, one would need to know its full binding profile to all cellular proteins. Savitski et al. take significant steps toward meeting this daunting challenge. They monitored the unfolding or “melting” of over 7000 human proteins and measured how small-molecule binding changes individual melting profiles. As a proof of principle, over 50 targets were identified for an inhibitor known to bind a broad spectrum of kinases. Two cancer drugs, vemurafib and Alectinib, are known to have a side effect of photosensitivity. The thermal profiling approach identified drug-protein interactions responsible for these side effects.

Science, this issue 10.1126/science.1255784

Abstract

The thermal stability of proteins can be used to assess ligand binding in living cells. We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine. We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL. Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity.

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