Abstract
The identification of protein targets and the elucidation of the molecular mechanism of action (MMoA) of bioactive small molecules are central goals of chemical biology. Many different techniques for target identification and engagement are developed, but none of them is generic. Here we describe one of these techniques—the cellular thermal shift assay (CETSA). The assay works without any labeling of proteins or small molecules, which allows the investigation of the unaltered interaction between the interaction partners. Briefly, the influence of small molecules on the thermal stability of proteins within whole cell lysates is investigated. We describe this approach in two variants: the conventional immunoblot-based approach (CETSA), as well as an unbiased approach based on a proteome-wide mass spectrometric analysis (thermal proteome profiling, TPP). The CETSA approach requires preknowledge about possible target proteins and can only detect a few proteins at once. Although TPP is technically more demanding, it allows for the identification of multiple (off)targets without any preknowledge.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ziegler S, Pries V, Hedberg C, Waldmann H (2013) Target identification for small bioactive molecules: finding the needle in the haystack. Angew Chem Int Ed Engl 52(10):2744–2792. https://doi.org/10.1002/anie.201208749
Schurmann M, Janning P, Ziegler S, Waldmann H (2016) Small-molecule target engagement in cells. Cell Chem Biol 23(4):435–441. https://doi.org/10.1016/j.chembiol.2016.03.008
Kapoor S, Waldmann H, Ziegler S (2016) Novel approaches to map small molecule-target interactions. Bioorg Med Chem 24(15):3232–3245. https://doi.org/10.1016/j.bmc.2016.05.020
Schenone M, Dancik V, Wagner BK, Clemons PA (2013) Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol 9(4):232–240. https://doi.org/10.1038/nchembio.1199
Lee H, Lee JW (2016) Target identification for biologically active small molecules using chemical biology approaches. Arch Pharm Res 39(9):1193–1201. https://doi.org/10.1007/s12272-016-0791-z
Kanoh N, Honda K, Simizu S, Muroi M, Osada H (2005) Photo-cross-linked small-molecule affinity matrix for facilitating forward and reverse chemical genetics. Angew Chem Int Ed Engl 44(23):3559–3562. https://doi.org/10.1002/anie.200462370
Lomenick B, Hao R, Jonai N, Chin RM, Aghajan M, Warburton S, Wang JN, Wu RP, Gomez F, Loo JA, Wohlschlegel JA, Vondriska TM, Pelletier J, Herschman HR, Clardy J, Clarke CF, Huang J (2009) Target identification using drug affinity responsive target stability (DARTS). Proc Natl Acad Sci U S A 106(51):21984–21989. https://doi.org/10.1073/pnas.0910040106
Savitski MM, Reinhard FB, Franken H, Werner T, Savitski MF, Eberhard D, Martinez Molina D, Jafari R, Dovega RB, Klaeger S, Kuster B, Nordlund P, Bantscheff M, Drewes G (2014) Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346(6205):1255784. https://doi.org/10.1126/science.1255784
Martinez MD, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y, Nordlund P (2013) Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341(6141):84–87. https://doi.org/10.1126/science.1233606 [doi] 341/6141/84 [pii]
Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ Jr, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund P, Sundstrom M, Weigelt J, Edwards AM (2006) Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A 103(43):15835–15840
Franken H, Mathieson T, Childs D, Sweetman GMA, Werner T, Togel I, Doce C, Gade S, Bantscheff M, Drewes G, Reinhard FBM, Huber W, Savitski MM (2015) Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat Protoc 10(10):1567–1593. https://doi.org/10.1038/nprot.2015.101
Reinhard FB, Eberhard D, Werner T, Franken H, Childs D, Doce C, Savitski MF, Huber W, Bantscheff M, Savitski MM, Drewes G (2015) Thermal proteome profiling monitors ligand interactions with cellular membrane proteins. Nat Methods 12(12):1129–1131. https://doi.org/10.1038/nmeth.3652
Huber KV, Olek KM, Muller AC, Tan CS, Bennett KL, Colinge J, Superti-Furga G (2015) Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling. Nat Methods 12(11):1055–1057. https://doi.org/10.1038/nmeth.3590
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. https://doi.org/10.1038/nbt.1511
Jafari R, Almqvist H, Axelsson H, Ignatushchenko M, Lundback T, Nordlund P, Martinez Molina D (2014) The cellular thermal shift assay for evaluating drug target interactions in cells. Nat Protoc 9(9):2100–2122. https://doi.org/10.1038/nprot.2014.138
Mateus A, Maatta TA, Savitski MM (2016) Thermal proteome profiling: unbiased assessment of protein state through heat-induced stability changes. Proteome Sci 15:13. https://doi.org/10.1186/s12953-017-0122-4
Instructions–TMT10plex mass tag labeling kits and reagents. Pierce biotechnology. https://tools.thermofisher.com/content/sfs/manuals/MAN0016969_2162457_TMT10plex_UG.pdf
Acknowledgment
We thank Dr. Marc SchĂĽrmann for the establishment of the TPP assay in our department. Without his large effort, it would not have been possible to perform the described assay in-house.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Reckzeh, E.S., Brockmeyer, A., Metz, M., Waldmann, H., Janning, P. (2019). Target Engagement of Small Molecules: Thermal Profiling Approaches on Different Levels. In: Ziegler, S., Waldmann, H. (eds) Systems Chemical Biology. Methods in Molecular Biology, vol 1888. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8891-4_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8891-4_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8890-7
Online ISBN: 978-1-4939-8891-4
eBook Packages: Springer Protocols