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The cellular thermal shift assay for evaluating drug target interactions in cells

Abstract

Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with experimental data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. One approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. Both approaches can be completed in a day.

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Figure 1: Schematic illustration of CETSA melt curve and ITDRFCETSA procedures using samples of different origins.
Figure 2: The screen format assay procedure.
Figure 3: Illustration of the remaining amount of p38α accessible for detection as a function of the temperature used for heat treatment of HL-60 cells.
Figure 4: Illustration of the amount of stabilized soluble p38α accessible for detection in the presence of increasing concentrations of compounds in HL-60 cells.
Figure 5: Scatterplot of the screening data after conversion to fold signal increase compared with the DMSO-only controls on each plate.

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Acknowledgements

D.M.M., R.J., M.I. and P.N. acknowledge Karolinska Institutet (Distinguished Professor Award), the Swedish Research Council (Vetenskapsrådet) and the Swedish Cancer Society (Cancerfonden). T.L., H. Almqvist and H. Axelsson acknowledge Karolinska Institutet and the Swedish Research Council, which funds Chemical Biology Consortium Sweden, a national infrastructure for research in the field of chemical Biology, as well as Biovitrum for a generous donation of instruments and compound libraries when Chemical Biology Consortium Sweden was inaugurated. We are also grateful to C. Zaniol at PerkinElmer for great support in the development of the AlphaScreen assays.

Author information

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Authors

Contributions

R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and D.M.M. conceived the study; R.J., H. Axelsson, H. Almqvist, T.L. and D.M.M. designed the experiments and the high-throughput formatting of the CETSA procedure; western blot and the AlphaScreen experiments were conducted by R.J., D.M.M., H. Axelsson, H. Almqvist and T.L.; and the manuscript was prepared by R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and D.M.M.

Corresponding authors

Correspondence to Thomas Lundbäck or Pär Nordlund or Daniel Martinez Molina.

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Competing interests

D.M.M. and P.N. are the founders of Pelago Bioscience AB. The remaining authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cellular integrity after heating.

In order to evaluate the integrity of the cell membrane of HL-60 cells during heating a trypan blue dye exclusion experiment was performed. Aliquots of approximately 3.0x104 HL-60 cells in culture medium were heated to 37, 50, 60, 65 and 70°C respectively for 3 min in the presence of 0.2% (w/v) trypan blue dye and allowed to cool to room temperature for 3 minutes. A 10μl aliquot of the heated cell suspension was subsequently analyzed using a TC20™ automated cell counter. Cells with the ability to exclude trypan blue were considered as retaining their cell membrane integrity whereas dye stained cells were identified as cells with disrupted cell membranes. The data demonstrated that the cell membrane of the HL-60 cells remain intact during heating at 50°C (the temperature at which the ITDRFCETSA experiments were conducted) since no statistically significant decrease in dye exclusion was observed compared to the initial dye exclusion capability (control) of the HL-60 cells. In addition, the data demonstrated that the HL-60 cell membranes are not disrupted unless temperatures above 60°C are reached. This is in agreement with our previous study where several other cell lines demonstrated loss of cell membrane integrity at temperatures above 60°C1.Data are given as the average ± standard deviation (n ≥ 4).

Supplementary Figure 2 Chemical structures.

The chemical structures of the p38α inhibitors; AMG-548 (A), SB203580 (B) and the dual Erk1/2 inhibitor ERK 11e (C).

Supplementary Figure 3 Negative control compound on p38α.

Western blot image illustrating remaining amounts of p38α in the presence of a dual Erk1/2 inhibitor, ERK 11e, in HL-60 cells as a function of temperature. All experiments were performed at three independent occasions and the experiments loaded and assayed in chorus using Western blot.

Supplementary Figure 4 ITDRFCETSA confirmation of CBK derived hit compounds.

An ITDRFCETSA experiment involving the human kinase p38α was performed to verify intracellular target binding of CBK200177 and CBK107148, both of which were identified in Box 4 (High throughput screening procedure for identification of stabilizers of intracellular p38α). Data are presented as the fold increase in AlphaScreen signal for each concentration of compound compared to DMSO controls. The data demonstrate dose dependent thermal stabilization of p38α for both compounds, thus confirming the results from the “High throughput screening procedure for identification of stabilizers of intracellular p38α”.

Supplementary information

Supplementary Figure 1

Cellular integrity after heating. (PDF 176 kb)

Supplementary Figure 2

Chemical structures. (PDF 224 kb)

Supplementary Figure 3

Negative control compound on p38α. (PDF 233 kb)

Supplementary Figure 4

ITDRFCETSA confirmation of CBK derived hit compounds. (PDF 260 kb)

Supplementary Data

Positive control compound on ERK1/2. (PDF 445 kb)

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Jafari, R., Almqvist, H., Axelsson, H. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc 9, 2100–2122 (2014). https://doi.org/10.1038/nprot.2014.138

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