Hot Topics: Cellular thermal shift assays to measure ligand-to-target engagement

The cellular thermal shift assay (CETSA) was introduced in July of 2013 as a means to investigate drug target engagement inside live cells and tissues (1). The underlying principle of CETSA is simple – it relies on the thermostability of each investigated protein and how this is altered by ligand binding. Experimentally these changes are assessed by applying a transient heat-pulse to the samples. This results in rapid rearrangements of established equilibria such that proteins denature and aggregate unless stabilised by ligand (1,2). The simplicity of CETSA has allowed prompt adoption in the literature but the importance of rapid changes in ligand binding is still not well recognised.

To explore these considerations we systematically varied both the heat-pulse temperature and duration in CETSA using p38a as our model system (3). Studies spanning seven different heating times and over a 13°C temperature interval show apparent potency changes over four orders of magnitude. These studies demonstrate how quantitative comparisons with functional cellular data require an understanding of the temperature dependence of the interactions under study. Our publications also discuss critical technology developments that allow shorter heating times.  These can now be down in the 10s of seconds range to minimize ligand rearrangements and heat-induced changes to cell permeability.

Comments by Dr. Thomas Lundbäck,  Associate Director, Mechanistic Biology & Profiling, Discovery Sciences, AstraZeneca R&D, Gothenburg,  Sweden,  thomas.lundback@astrazeneca.com. 

1. Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P., Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 2013, 341 (6141), 84-7 (PMID 23828940).
2.  Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Martinez Molina, D., The cellular thermal shift assay for evaluating drug-target interactions in cells. Nat Protoc 2014, 9 (9), 2100-22 (PMID 25101824).
3.  Seashore-Ludlow, B.; Axelsson, H.; Almqvist, H.; Dahlgren, B.; Jonsson, M.; Lundbäck, T., Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay. Biochemistry Nov 2018 (PMID 30418016).