Target Engagement Reagents
The pharmacological validation of the interaction of a ligand with its protein target is referred to as target engagement. Target engagement studies are crucial in drug discovery as they can provide confirmation that the compound(s) under study are indeed binding to the target protein at the intended site in order to be both effective and safe. Traditionally, target engagement has been assessed on purified proteins in buffer or deduced from downstream pathway effects such as post-translational modifications (PTMs) or enzyme substrate changes. Both of these approaches have severe limitations such as lack of a physiologically relevant environment in the case of purified proteins, or the inability to confidently attribute PTMs to one particular protein target due to the existence of multiple pathways controlling modification.
Even target engagement methodologies that do take place within a cellular environment such fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) technologies have limited physiological relevance, since they require modifications to the protein and cloning.
Why CETSA®
The Cellular Thermal Shift Assay (CETSA) is based on the change of the thermal denaturation profile of the native target protein in a cellular context that occurs following the binding of a ligand (i.e. a drug candidate). CETSA is performed by incubating cells with the test compound, followed by heating of the compound-treated cells, and then by quantifying the remaining soluble target protein. The thermally stabilized protein-ligand complex can then detected by Alpha or HTRF® assay technology. Both technologies are no-wash, highly sensitive with a broad dynamic range, and provide fast time-to-results. CETSA is a patented method and a licence from Pelago Bioscience is required to use the technology.
CETSA can be combined with antibody pair screening technologies such as Alpha or HTRF allowing the study of thousands of compounds in a High-Throughput fashion.
CETSA combined with a proximity-based assay such as Alpha or HTRF:
- Is a cell-based, physiologically relevant target engagement study tool
- Uses endogenous protein levels in unmodified or even primary cells
- Can be applied throughout the drug discovery workflow in a reliable, high-throughput format
Alpha / HTRF CETSA is a real game changer in that it combines an intracellular, physiologically relevant assay with high-throughput potential and amenability to automation.
CETSA Validated vs CETSAble®
Revvity and Pelago Biosciences currently offer fully validated Alpha CETSA kits covering 13 different protein targets, including EGFR, BCL-2, NFκB, and p38 MAPK.
We also recommend an additional 40 AlphaLISA® SureFire® kits for protein targets that have been pre-validated by Pelago as CETSAble. This pre-validation ensures that the protein target displays physical properties that make it amenable to thermal shift studies as well as the applicability of the Alpha assay in the context of CETSA.
CETSA is a patented method and the use of these kits with CETSA is permitted under license from Pelago Bioscience.
A CETSAble assay has undergone validation as to the amenability of the protein target to heat-based denaturation studies as well as the usability of the approach in common cell lines. It will require further optimization in the end-user’s hands for their particular application and experimental conditions.
Alpha
For results in half the time of ELISA, without wash steps, and with excellent sensitivity in a variety of matrices, including serum and plasma.
For results in half the time of ELISA, without wash steps, and with excellent sensitivity in a variety of matrices, including serum and plasma.
The principle of detection of the soluble protein by the Alpha CETSA® assay is shown in the figure above. Only the soluble protein will lead to the generation of an Alpha signal (top), while the thermally denaturated insoluble protein does not generate the proximity needed for an Alpha signal (bottom).
Alpha SureFire
For studying pathways or phosphorylated proteins in whole cells or studying endogenous receptors.
For studying pathways or phosphorylated proteins in whole cells or studying endogenous receptors.
The principle of detection of the soluble protein by the Alpha SureFire CETSA® assay is shown in the figure above. Only the soluble protein will lead to the generation of an Alpha signal (top), while the thermally denaturated unsoluble protein is unable to create the proximity needed for the generation of an Alpha signal (bottom).
How can we help?
When it comes to developing assays or troubleshooting issues, always know you’re never on your own.
When it comes to developing assays or troubleshooting issues, always know you’re never on your own.
Whatever the assay you have in mind, you need the right technologies for your target. We can custom conjugate your antibody or biomolecule to any bead, or biotinylate or DIG-label your targets, so you can develop the assay you need. If you prefer, we can even perform the assay development for you.
FAS: Our global team of Field Application Scientists are hands-on subject matter experts in assay development. They are available to assist in both assay development and troubleshooting.
Application support knowledgebase (ASK): Need help at 1am? We’ve got it covered. The Application Support Knowledgebase (ASK) contains detailed information on assay development, protocols, tips, FAQs, citations, troubleshooting and more. Accessible 24 hours a day, 7 days a week.
