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

Nature Methods volume 12, pages 1055–1057 (2015)Cite this article

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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.

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Figure 1: Schematic representation of the thermal profiling methodology.
Figure 2: Thermal profiling results for the MTH1 inhibitor (S)-crizotinib, MTX and 2′3′-cGAMP.

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Acknowledgements

We are grateful to W. Berger (Institute of Cancer Research, Vienna, Austria) for providing SW480 cells and P. Majek (CeMM, Vienna, Austria) for assistance with data processing. This work was supported by the Austrian Academy of Sciences, the European Union (FP7 259348, ASSET) and the Austrian Science Fund (FWF F4711, MPN).

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Author notes

  1. Kilian V M Huber, Chris Soon Heng Tan & Jacques Colinge

    Present address: Present addresses: Structural Genomics Consortium, University of Oxford, Oxford, UK and Target Discovery Institute, University of Oxford, Oxford, UK (K.V.M.H.); Institute of Molecular & Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore and Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore (C.S.H.T.); Institut de Recherche en Cancérologie de Montpellier, Inserm U1194, Université Montpellier, Montpellier, France (J.C.).,

Authors and Affiliations

  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

    Kilian V M Huber, Karin M Olek, André C Müller, Chris Soon Heng Tan, Keiryn L Bennett, Jacques Colinge & Giulio Superti-Furga

  2. Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

    Giulio Superti-Furga

Authors
  1. Kilian V M Huber
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Contributions

K.V.M.H. designed experiments and jointly performed them with K.M.O.; A.C.M. and K.L.B. performed mass spectrometry; C.S.H.T. and J.C. performed bioinformatics analysis; and K.V.M.H. and G.S.-F. conceived the study and wrote the manuscript. All authors contributed to the discussion of results and participated in preparation of the manuscript.

Corresponding authors

Correspondence to Kilian V M Huber, Jacques Colinge or Giulio Superti-Furga.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Data normalization.

Average thermal profiles are plotted for DMSO- and (S)-crizotinib-treated cells. Note that the two profiles (broken straight lines) are similar (no thermal shift) but imperfect. The two fitted sigmoid models are very close (dashed smooth lines), thus further indicating the absence of influence of (S)-crizotinib on the average thermal stability. Vertical arrows (only displayed for the DMSO sample for simplicity), expressed as shift in normalized coordinates, can be applied to spectra specific to a given protein for subsequent scoring of the whole dataset and can be regarded as a structural normalization considering the different protein content at each temperature.

Supplementary Figure 2 Known interactions between proteins found to be stabilized in experiments with intact SW480 cells.

Edge thickness represents interaction confidence. Data and graphical representation taken from STRING1.

Supplementary Figure 3 Original full-length anti-MTH1 western blots used for Figure 2b.

Data from SW480 intact cell experiments. (a) DMSO; (b) (S)-crizotinib. Asterisk indicates unspecific band.

Supplementary Figure 4 Melting-temperature profiles obtained for MTH1 (NUDT1) from SW480 cell lysate experiments.

Similar to intact cell experiments, treatment of SW480 cell extracts with (S)‑crizotinib leads to a stabilization of MTH1 (NUDT1). Data indicate one technical replicate.

Supplementary Figure 5 Kinases with differential melting profiles observed in cell lysates treated with 100 µM (S)-crizotinib.

Treatment of SW480 cell lysates with 100 µM (S)‑crizotinib leads to a stabilization of (a) PI3K-beta (PIK3CB) and (b) CDK2. Data indicate one technical replicate.

Supplementary Figure 6 Original full-length anti-DHFR western blots used for Figure 2d.

Data from K562 intact cell experiments. (a) DMSO; (b) methotrexate.

Supplementary Figure 7 Original full-length anti-STING western blots used for Figure 2f.

Data from RAW intact cell experiments. (a) Water; (b) 2ʹ3ʹ-cGAMP.

Supplementary Figure 8 Definition of the area score.

The area score considers a signed area between the vehicle-treated sample curve and the compound-treated curve.

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Supplementary Figures 1–8, Supplementary Tables 1–5 and Supplementary Note 1 (PDF 2462 kb)

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Huber, K., Olek, K., Müller, A. et al. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nat Methods 12, 1055–1057 (2015). https://doi.org/10.1038/nmeth.3590

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