Binding of a ligand to the native three-dimensional configuration of a protein increases protein stability, with a corresponding increase in the protein’s melting temperature. This indirect measurement of ligand binding is measured in a thermal shift assay (TSA).1

Binding of compounds to a purified protein target can be measured using a range of biochemical or biophysical assays, such as differential scanning fluorimetry (DSF). However, these measures do not always translate into target engagement in a cellular context. In DSF, purified protein is mixed with an environmentally sensitive dye, whose fluorescence increases with the concentration of unfolded protein in the mixture. Fluorescence intensity of the dye is recorded as a function of temperature, resulting in a protein melt curve. Performed in the absence and presence of ligands, a thermal shift (ΔTm) is calculated.1

Cellular thermal shift assays

Ligand-induced thermal stabilization also occurs within the context of the intracellular environment.1,2 The Cellular Thermal Shift Assay (CETSA), a proprietary protocol developed by Pelago Bioscience, is an easy and proven method to demonstrate ligand-protein interactions in living cells and can be used without modifications to the ligand or protein. The gain-of-signal assay produces minimal false positives and can be applied throughout all steps of the drug discovery process.2

Conceptually not that different than traditional TSAs, CETSA can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns. To demonstrate how well CETSA correlates to more established and conventional cellular and biochemical TSA approaches Shaw et al. reported two novel high-throughput CETSA (CETSA HT) assays for protooncogene B-Raf, and the polymerase PARP1 (Shaw’s work was performed under license from Pelago). Comparative analyses with other assays showed that CETSA HT correlates well with other screening technologies.

CETSA datasets, especially when combined with other assay formats, can help build a much clearer picture of how targets modulate complex systems. In the assay, live cells are treated with test compounds, and a short transient heat shock is applied. The cells are then lysed and the levels of the thermostable target protein quantified.2

In another set of experiments, Shaw and team used western blots to show stability of the androgen receptor when bound in live cells. Western blots can be quantified to demonstrate the thermal shift as evidence of target engagement. But homogenous bead-based approaches such as PerkinElmer Alpha CETSA technology can also be applied to quantify the thermally stable target protein.4 This high-throughput assay can be used to quantify structure activity relationship (SAR) against an entire chemical series to correlate to other datasets.2

 In vitro examples

In a quantitative approach, cells can be treated with a range of concentrations of the test compound. A single heat shock is then applied at a fixed temperature to all samples to measure a relative EC50 as an estimate of the potency of the binding of the target in the cells.2

PARP1 is a well-established target across a range of different cancers. When a relevant cell background was treated with different PARP1 inhibitors and an alpha endpoint was used to quantify PARP1, a thermal shift showed evidence of target engagement but did not differentiate the inhibitors. They all bound PARP1. By performing an isothermal dose response with a 49°C heat shock, a measure of potency was determined for these compounds and measured as an EC50 to differentiate the compounds based on the potency with which they bound PARP1.2, 3 Importantly, the results showed where binding of protein does not lead to binding in cells and when binding in cells does not lead to functional inhibition of the target.2

Another example uses ROS1, a receptor tyrosine kinase that is involved in chromosomal rearrangements on fusion proteins and responsible for driving subsets of non-small cell lung cancer. ROS1 can form a range of different fusions where the fusion proteins become joined to another protein. A range of inhibitors against this fusion were explored.2 Activity was measured against the isolated kinase domain and functional activity against the fusion protein within the HCC78 cell background.

Using Alpha CETSA datasets, the researchers demonstrated that binding to the fusion protein translated to binding to the protein in cell lysates and in turn, live cells. But correlations can be less clear—activity in cells and target engagement may be different compared to assays on purified protein.2

References

    1. Shaw J and Stubbs C. Indirect Detection of Ligand Binding by Thermal Melt Analysis. Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, 2021, Methods in Molecular Biology, vol. 2263, DOI:10.1007/978-1-0716-1197-5_8
    2. Cellular Thermal Shift Assays – bringing relevant target engagement to your drug discovery workflow webinar. Sponsored by PerkinElmer and Pelago Bioscience. Drug Target Review. 2021. https://www.drugtargetreview.com/webinar/90745/cellular-thermal-shift-assays-bringing-relevant-target-engagement-to-your-drug-discovery-workflow/
    3. Shaw J, et al. Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1. SLAS Discovery 2019, Vol. 24(2) 121–132. DOI: 10.1177/2472555218813332
    4. Shaw J, et al. Determining direct binders of the Androgen Receptor using a high throughput Cellular Thermal Shift Assay SCIENTIFIC REPORTS, 2018, 8:163, DOI:10.1038/s41598-017-18650-x