Abstract 1848: The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker discovery

Abstract With the Cellular Thermal Shift Assay (CETSA) celebrating its first decade since the PoC publication, the method has gained much interest in the basic science field as well as become an industry standard in applied drug discovery setting. In the last few years it has become apparent that in addition to providing information about drug-protein interaction, CETSA based thermal profiling of cells can help describe additional aspects of cell biology as it gives insight into the activating and rewiring of protein-protein interaction networks inside the cell upon stimuli. In the work presented here, we have used CETSA coupled to high resolution mass spectrometric readout to profile the response patterns of about 8000 proteins to several hundred molecular probes and marketed drugs. The goal has been to provide a map of wanted and unwanted effects following treatment and to correlate it to alternative readouts, by for example cellular imaging and cell death assays. We have applied the CETSA protocol for close to five hundred compounds, many in several cell-lines (HepG2, U87MG and K562). The experimental setup includes the step of treating the cells with a certain compound at a fixed concentration. After incubation for an hour, we heated the cells to a range of temperatures, followed by a lysis protocol and pooling of the samples. After this, the samples have been centrifuged to separate the soluble from insoluble fraction. The soluble fraction has then been prepared for MS acquisition using TMT based multiplexing. Typically, this allowed us to monitor changes in thermal stability among more than 8, 000 proteins, as a consequence of compound treatment. This data reveals specific compound “fingerprints” that can be used to decipher mode of action and identify biomarkers. Despite the short incubation time, proteins with compound-induced thermal stability shifts are not only targets/off-targets (direct binders), but also downstream, and sometimes upstream, pathway members and general cellular responses. All data combined makes the basis of the Target Engagement Atlas. Here we can see clusters of compounds, sharing similar protein binding profiles. Among the most prominent ones are mTOR inhibitors, NSAIDs, as well as tubulin binders. Alternatively, one can use this resource to reveal clusters of “pharmacologically” associated protein networks. We show that pharmacological perturbation allows identification of clusters representing tight protein complexes (for example ribosomes), known metabolic pathways (folate biosynthesis), molecular functions (kinases), as well as networks of proteins linked only by ligand binding specificity. CETSA MS allows for unbiased, cellular, and molecular profiling of compound effects. Together with data from clinical settings, as well as in vivo/in vitro end point assays it is possible to correlate the CETSA patterns to wanted but also unwanted responses. Citation Format: Tomas Friman, Alexey Chernobrovkin, Tuomas Tolvanen, Erin Gilson, Stina Lundgren, Victoria Brehmer, Daniel Martinez Molina. The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1848.