Target Engagement of Small Molecules: Thermal Profiling Approaches on Different Levels
The identification of protein targets and the elucidation of the molecular mechanism of action (MMoA) of bioactive small molecules are central goals of chemical biology. Many different techniques for target identification and engagement are developed, but none of them is generic. Here we describe one of these techniques—the cellular thermal shift assay (CETSA). The assay works without any labeling of proteins or small molecules, which allows the investigation of the unaltered interaction between the interaction partners. Briefly, the influence of small molecules on the thermal stability of proteins within whole cell lysates is investigated. We describe this approach in two variants: the conventional immunoblot-based approach (CETSA), as well as an unbiased approach based on a proteome-wide mass spectrometric analysis (thermal proteome profiling, TPP). The CETSA approach requires preknowledge about possible target proteins and can only detect a few proteins at once. Although TPP is technically more demanding, it allows for the identification of multiple (off)targets without any preknowledge.
Key wordsProteomics Target identification Target engagement (Off)target Cellular thermal shift assay (CETSA) Thermal proteome profiling (TPP) Immunoblot Nano-LC-MS/MS
We thank Dr. Marc Schürmann for the establishment of the TPP assay in our department. Without his large effort, it would not have been possible to perform the described assay in-house.
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