Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling

Abstract

Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2'3'-cGAMP in intact cells and identified the 2'3'-cGAMP cognate transmembrane receptor STING, involved in immune signaling.

Figure 1

Schematic representation of the methodology. (a) Binding of a ligand to its target protein (top) increases the enthalpy required for unfolding. As a result the melting temperature (T_m) is shifted which can be exploited by e.g. differential scanning fluorimetry (DSF, “Tm shift assay”) (bottom). (b) Representative workflow. (c) Schematic illustrating the bioinformatic scoring parameters used for data analysis. Continuous lines depict raw data, dashed lines are curve fitting results.

Figure 2

Thermal profiling results for the MTH1 inhibitor (S)-crizotinib, methotrexate (MTX), and 2′3′-cGAMP. (a) Protein abundance graphs obtained from MS-TMT-10-plex experiments. In contrast to the cognate target MTH1 (NUDT1), the close family member NUDT5 is not stabilized upon treatment of intact cells with the specific inhibitor (S)-crizotinib. Data indicate two independent experiments (n = 2). (b) Western blot confirming the stabilization of MTH1 protein by (S)-crizotinib as compared to DMSO using intact SW480 cells. Images are cropped for clarity; full-length blots are presented in Supplementary Figure 3. (c) Protein abundance graphs obtained from MS-TMT-10-plex experiments demonstrating a strong shift in thermal stability for the well-established MTX target DHFR. Data indicate two independent experiments (n = 2). (d) Immunoblot confirming the stabilization of DHFR by methotrexate (MTX) compared to DMSO using intact K562 cells. Images are cropped for clarity; full-length blots are presented in Supplementary Figure 6. (e) MS analysis results of RAW macrophages treated with 2′3′-cGAMP reveal stabilization of the transmembrane receptor STING (Tmem173). Data indicate two independent experiments (n = 2). (f) Western blot validation of the increased thermal stability observed for STING after treatment with 2′3′-cGAMP. Images are cropped for clarity; full-length blots are presented in Supplementary Figure 7.