CETSA® by Pelago Bioscience is a well-proven, best-in-class technology used to assess cellular protein-ligand binding. CETSA® can be applied throughout all steps of the drug discovery value chain. Because neither the ligand nor the protein is modified, CETSA® provides reliable measurements of target engagement in authentic cellular environments and under actual physiological conditions. In this way, biologically relevant values can be obtained from any type of cellular matrix – i.e. cell lines, animal tissue or patient samples – to help you explore, discover and confirm drug candidates faster.
How does CETSA® work?
The CEllular Thermal Shift Assay (CETSA®) is based on the same biophysical principle as traditional thermal shift assays (TSAs), but leverages the novel insight that melt curves of proteins can also be measured in intact cells and that proteins unfold and aggregate at individual and specific temperatures. The very basis for thermal shift assays, cellular or not, is that the thermal stability of proteins can change if their environment is altered.
In its simplest form, CETSA® is carried out on a single protein basis, assessing target engagement qualitatively by incubating and heat shocking cells in presence and absence of the studied compound. Quantifying the amount of protein that remains soluble after heat shock and plotting this to a range of different temperatures gives the CETSA® melt curve of the protein. If the compound has bound to the target protein the latter becomes more or less resistant to heat, causing a shift in the melt curve (also known as a thermal shift).
Target engagement can also be quantified by exposing the cells to different concentrations of the compound and comparing the extent of stabilization of the target protein at specific temperatures. Such concentration-response experiments allow CETSA® specific potencies of target engagement to be determined. These potencies incorporate more than just the protein-ligand affinity, they also include factors such as membrane permeability, cellular activation or degradation, accessibility of the protein binding site…all the factors that make data generated within a cellular system more valuable.
Naturally, this gives you more accurate and biologically relevant assessment of target engagement compared to traditional biochemical or molecular biology methods that require tailored cellular systems, tagged or truncated proteins in highly defined buffers or modified compounds.
Below you can read and find information about the different CETSA® formats, all fit for purpose and based on the same underlying principles.
CETSA® - Flexible assay formats
CETSA® Navigate (formerly known as Classics) is a targeted method applied to single selected proteins. Detection is based on Western Blot and is suitable for studies examining 1-10 compounds at a time. This format is well-suited for early feasibility studies, hit confirmation, lead optimization, and translational work to progress your project to the next phase. At Pelago, such projects are carried out routinely allowing rapid execution, from project start to final report the expected timeline is 8-10 weeks.
Read more in our Target Engagement eBook.
CETSA® Navigate MS
CETSA® Navigate MS is a complementary targeted method that allows you to pursue target proteins where no antibodies are available for detection. Quantification is based on Mass spec and uses trace peptides as internal standards allowing for specific and robust profiling of individual or multiple proteins.
CETSA® Navigate MS is a valuable method to investigate different isoforms or to perform selectivity studies. Same as with the CETSA® Navigate method, this format can be applied for studies examining 1-10 compounds at a time in early feasibility studies, hit confirmation, lead optimization, and translational work. From the project start to the final report the expected timeline is around 8-10 weeks.
CETSA® Navigate HT
CETSA® Navigate HT was developed to increase the throughput of our targeted method. We typically use dual antibody luminescence-based detection systems that allow plate-based automation. Navigate HT is ideal for library screening, hit confirmation and lead optimization. Typically, an assay development phase of 8-10 weeks and is followed by a screening phase with a throughput of 10-50,000 data points per week.
Read more in our Target Engagement eBook.
CETSA® Explore, also known as TPP (Thermal Proteome Profiling) or PISA (Protein Integral Solubility Alteration), utilizes a proteome-wide mass spectrometry-based detection method. It can analyze the systemic effect of a compound on up to 6,000 key proteins in a single experiment. Measuring not just the direct binding of compound to proteins but also the subsequent downstream consequences of the initial target engagement. This unbiased proteome wide profiling makes it ideal for Mechanism-of-Action (MoA) investigation, as well as for phenotypic target deconvolution and safety assessments. Once the experimental parameters have been specified, the turnaround time is usually 10-12 weeks depending on the project scope.
Proteins of interest uncovered by CETSA® Explore are amenable to both Navigate and Navigate HT methods. This allows for the generation of specific assays suitable for low or high throughput once targets have been identified.
Read more in our CETSA® Explore eBook.
CETSA® can determine cellular target engagement in physiologically relevant settings of the native, full-length protein and can potentially unlock novel chemical space.
CETSA® can assess target engagement across a multitude of sample types. Established assays can easily be transferred from cell line to animal tissue, human primary cells, or patient–derived material, and thereby be used for translation all the way from lab bench to patient.
Since the protein targets of any given compound hit from a phenotypic screen are initially unknown, one major challenge with phenotypic–based drug discovery is the need for target identification or deconvolution.
Biomarkers are important to facilitate translation, validate the therapeutic hypothesis and monitor therapeutic effect in clinical trials and beyond. The identification of biomarkers is a time–demanding but crucial part of the preclinical drug discovery process
The aim at this stage of the drug discovery and development process is to optimize the compound properties to ensure the development of safe and effective candidate drugs (CDs) that can be dosed in healthy volunteers.
In drug discovery, selective binding of the compound to the right target is important for the drug to be both effective and safe. To achieve this, the drug must be present at the site of action and occupy the intended target with high specificity.
Access our resource library of downloadable content. Explore our eBooks, application notes and additional useful information.
Use our dedicated resource library and knowledge center to learn more. Contact us to confirm your hypothesis on how to apply CETSA® to your projects.
Learn more about how the CETSA® technology can be applied and the value it provides in different stages of the drug discovery and development process.