The Cellular Thermal Shift Assay is a method that allows the quantification of a compound’s Target Engagement (TE) within living cells or in disrupted cells. The CETSA® assay principle is based on the change in thermal denaturation profile of the target protein that occurs following the binding of a compound. The CETSA® assay is performed by incubating the cells with the test compound, followed by heating of the compound-treated cells, and then by measuring the remaining soluble target protein. Read more about CETSA®.
In the absence of confirmation that a compound indeed interacts with the desired target, drug developers can lose precious time and resources moving in the wrong direction. For that reason, confirmation of a compound’s Target Engagement has long been regarded as essential in drug discovery. Such assays enable direct comparisons of a compound’s affinity for its target to be made, as such it is an essential measure to select which compounds to advance at all stages of lead generation and optimization. Because Target Engagement can be correlated to affinity it enables researchers to select compounds that have the highest affinity values. As such it is an extremely useful measure to ensure the correct compound prioritization at each stage of the drug discovery process.
Beside CETSA®, there are numerous methods for assessing Target Engagement (e.g. Surface Plasmon Resonance, Fluorescence Polarization and Thermal Shift). However all these methods rely on purified protein being available and can only be applied in vitro, meaning the derived affinity values may not be predictive of actual affinity or binding potential in living cells. Being able to run Thermal Shift assays in a relevant cellular context, where the protein is in its native environment, in the presence of its natural partner proteins, with physiological concentrations of its cofactors and substrates, yields much more relevant data, that will better translate into animal models and clinical trials.
While the Cellular Thermal Shift Assay is a method based on the same biophysical principal as standard Thermal shift assays (i.e. that specific proteins will denature at a set temperature) it does not directly measure the specific temperature of unfolding, but relies instead on the fact that the presence of a compound on the protein will affect the amount of soluble protein present after heating to a set temperature. As such, CETSA® is in essence a total protein assay conducted after a specific heating event. Compounds with differing target engagement potencies will change the relative amounts of protein surviving the heating event. Because the assay measures this residual protein, rather than actual melting event, it can be applied to more complex in vivo systems such as cells and lysates (and in some CETSA® formats even solid tissue). In addition, CETSA® can identify compounds that destabilize a protein (i.e. reduce its melting temperature), this is less straight forward with the other thermal shift methods
Yes, we have run many CETSA® Navigate projects, in western blot, with animal tissue dosed samples, to assess target engagement in tissue.
The client can send the dosed tissue or we can prepare the samples with a collaborator.
Depending on the target and the scope of the project, it might be useful to develop the assay of target engagement in a relevant cell line first, with melt curves and concentration responses, then continue with an assay development in tissue extract and follow in vivo dosed animal studies for target engagement quantification experiments.
The altered thermal stability of a protein upon ligand binding can result in either stabilization or destabilization of the protein and (both) are indication of cellular target engagement.
Destabilizations can be caused, for example, by the compound interfering with a protein-protein interaction and thereby disrupting of a protein complex. Another example can be when the compound competes with the natural substrate, for example ATP.
In our experience, membrane proteins usually have a higher melting temperature and are more often destabilized than stabilized by compound binding, and the preferred experimental matrix is the intact cell where the protein is in its native environment embedded within the membrane.
There is not a limit for confirmation of target engagement, very small shifts of less than one degree can confirm target engagement as long as the shifts are stable and reproducible, with several biological repeats. Large proteins with high intrinsic thermal stability tend to yield very small shifts and need normally higher compound concentration for the shift to be resolved.
CETSA®-derived XC50, from a concentration response assay, can be correlated with different Kd affinity assays. An in house example was performed with CETSA®-derived XC50s in CETSA® Explore, where concentration response curves were fitted from 5 concentration points with the pan-kinase inhibitor Staurosporine in K562 cells. The XC50 values were then correlated with published Kd values from competitive binding assays on synthetic kinases. (Chernobrovkin et al BioRviX 2020 https://doi.org/10.1101/2020.03.13.990606)
The CETSA® method can resolve potencies from fM to mM. In order to establish a melt and shift curve with a saturating concentration of a low potency compound, it need to have a very good solubility in saline buffers. As a rule of thumb we suggest to test at least 10x the expected XC50 concentration.
We typically observe a log unit right shift of CETSA® EC50s (cellular target engagement) potencies versus XC50 potencies from biochemical affinity assays, and in similar range from other cellular assays, although this will depend on the target, compound and read out system.
As mentioned above, CETSA® is basically a total protein assay conducted after heat shock. Alpha CETSA® uses a dual antibody proximity based detection system. Other methods avoid the use of an antibody based system by modifying the target protein:
- Promega NanoBRET Target Engagement Assay : Luciferase Fusion Tag combined with labelled tracer compounds which, when in close proximity to the luciferase will lead to Bioluminescence Resonance Energy Transfer (BRET). Compound binding in the same pocket will displace the tracer and BRET signal will decrease.
- Promega nanoluciferase thermal shift assay (NaLTSA) : Nanoluc Luciferase Fused to the target protein (recombinantly expressed protein); When the protein target with the Nanoluc enzyme tag aggregates, the luciferase signal will decrease.
- DiscoverX InCELL Pulse : Based on Enzyme Fragment Complementation (EFC) technology : the target protein is fused with a small enzyme donor fragment of β-galactosidase (β-gal). When the active enzyme hydrolyzes a substrate a chemiluminescent signal is generated and protein abundance can be quantified.
The key difference between these “recombinant CETSA® or “recombinant target engagement” systems and Alpha CETSA®, is that they cannot be applied to native and unmodified cells. This has a number of negative consequences:
- While the cost of developing a novel antibody pair and the subsequent per well costs may be higher than simply transfecting a single cell line, it is likely that any discovery program will want to be tested with a variety of model cell lines, each transfection comes with its own costs and cloning out the subsequent cell lines will take additional weeks.
- Generating a tagged protein will always have physiological consequences on the cell and the tagged protein may be (i) overexpressed (wrong stoichiometry vs. partners), (ii) not located in the correct cellular compartment or (iii) not associated with key partner proteins and (iv) may have a different melting behaviour than the native protein. Meaning that the apparent effect of a compound may not reflect its real behavior in patient tissue. These problems may be magnified in any temporary transfection system.
- Luciferase inhibitors may result in false positive hits.
- In later stages of lead optimization when more complex and physiologically more relevant cellular models (primary, native cell lines and tissue) are required, tagged protein approaches are simply not applicable.
CETSA® provides a unique measure of a compounds ‘on target presence’ and can be thought of as target occupancy. This occupancy will be affected by both the compounds ability to engage the target and the ability of the compound to be present at the right location (i.e. does it have the right solubility, permeability, metabolic stability and availability at the right cellular compartment). Non-cellular Target Engagement assays offer measures of affinity that correlate only with the protein’s behavior in highly artificial environments often using purified recombinantly expressed target proteins.
Upon heating a protein will encounter a temperature at which it denatures (sometimes referred to as the melting point). This melting temperature is a physical property and a constant for any given set of conditions (pH, pressure, salts). Compounds that interact with a protein will change the melting temperature (thermal shift) of the target protein. There are a number of variations on the in vitro Thermal Shift assay, but the key technique was first described by Semisotnov et al. (1991). In this method, the melting and unfolding protein exposes hydrophobic surfaces that enable SYPRO orange to bind. This dyes’s fluorescence is quenched in water, so the binding to these hydrophobic surfaces reverses this and causes fluorescence to peak when the protein is fully unfolded. This type of Thermal Shift assay can only be applied to highly purified proteins and for low abundance species this may mean that an over expression system is required to generate sufficient amounts.
Thermofluor – temperature sensitive dye system (using Sypro Orange)
Differential scanning fluorimetry (DSF) – alternative dye based methods
Quantitive PCR systems (eg Roche Light cycler) : these are used to deliver the heating event and can measure fluorescence changes
Nanotemper – is a company manufacturing dedicated instrument that heats the sample and measures the fluorescence. It offers better performance than the adapted PCR systems.
Pelago has experience developing assays for target engagement of antibodies.
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