Case Story: CETSA for High Throughput Screening
Tackling a Challenging Target
A leading biopharmaceutical company set out to pursue a highly challenging disordered protein target. Several factors made this particularly complex: the target was part of a multi-protein complex, making in vitro biology difficult to reproduce; its conformational flexibility hindered traditional approaches; and, at the time, there were no known small molecule binders or inhibitors to use as positive controls for assay development. Previous work with this target underscored the need to study it in its relevant cellular environment, in the presence of natural interaction partners.
Solution: CETSA for Primary Screening
To overcome these challenges, the company partnered with Pelago Bioscience. Using CETSA, the team established an assay in intact cells, enabling the study of the target in its physiologically relevant context. Unlike recombinant protein assays or indirect pharmacodynamic readouts, CETSA directly measured compound binding through thermal stabilization, providing confidence that observed effects reflected cellular biology.
CETSA was integrated into the customer’s broader screening strategy, including biochemical ligand-binding assays, enzyme activity tests, and biophysical methods such as thermal shift assays on recombinant protein and fragment screening for structural studies. This enabled cross-comparison of data and validation of hits across multiple platforms.
Delivering Novel Chemical Scaffolds
Through a high-throughput screening campaign of ~50,000 chemically diverse small molecules, CETSA identified 14 compounds that shifted the protein’s melting temperature by more than 1°C. Importantly, other screening methods did not detect these scaffolds, demonstrating the unique value CETSA brought to the campaign. Signal-to-noise ratios remained robust, and the screening produced reproducible results with Z-factors averaging 0.6, underscoring assay reliability.
Hits were further confirmed in follow-up assays, enabling optimization and progression of the project. Based on the success of this approach, the customer has since extended the use of CETSA primary screening to larger compound libraries, opening new avenues for discovery on challenging targets.
STRATEGIC INSIGHTS: The project team highlighted the advantages of CETSA in enabling the team to interrogate a previously intractable target directly in cells. By working with Pelago Bioscience, they successfully identified novel chemical compounds that would have been challenging to identify through traditional screening methods. This partnership demonstrates how CETSA empowers drug discovery teams to pursue challenging targets with greater confidence and efficiency. CETSA provides insights that accelerate discovery and reduce risk in early R&D by enabling direct measurement of compound-target engagement in intact cells.
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