Abstract 408: CETSA-based evaluation of non-degraded PROTAC targets

Abstract Protein degradation is a non-classical modality with the potential to tackle therapeutically interesting proteins, even those previously deemed undruggable. The molecular effect that a productive interaction between target proteins and a bifunctional degrader/PROTAC can easily be monitored by measuring protein degradation. However, not all proteins that bind degraders are in fact degraded, which suggests that not all effects of PROTACs are based on the degradation of targets, but also the effects of traditional binary protein - ligand interactions. The Cellular Thermal Shift Assay (CETSA) is a powerful technology for identifying the interactions between compounds and their cognate protein targets. Coupling CETSA with mass spectrometric (MS) readout allows for profiling of binding selectivity of both the warhead that binds the protein of interest (POI) but also to investigate the selectivity for different PROTAC scaffolds. CETSA can be carried out in both lysates and intact cells, always without the need to modify the compound, the proteins or the cellular environment. In a typical CETSA workflow we can address both the degradation profile of proteins in the proteome that is accessible by MS, and also provide insights into the binding of the degrader to both the E3 ligase and the POI. This allows us to identify any disconnect between degradation and binding of degrader as well as to follow the downstream cellular and molecular effects. Here we will exemplify how CETSA can be applied in deconvoluting protein binding of a CDK9 targeting PROTAC: THAL SNS 032, which is composed of the cereblon (E3-ligase) binder Thalidomide and the CDK (POI) binder SNS 032. THAL SNS 032 induces degradation of the annotated target CDK9 and its associated protein Cyclin-T1, but target engagement can also be observed for additional proteins, including other kinases. In turn, the non-productive interactions from a degradation point of view can still result in biological effects, as exemplified here where GSK3A and GSK3B as well as their downstream substrate FOXK1 are thermally shifted but not degraded by the PROTAC. The effects on the GSK3 - FOXK1 signaling axis would have been missed if only protein degradation had been considered. By combining mass spectrometric data from CETSA experiments with degradation readouts it is possible to correlate cellular target engagement potencies with degradation efficiency and importantly, also identify any drug - protein binding that do not result in degradation Citation Format: Tomas Friman, Tuomas Tolvanen, Alexey Chernobrovkin, Daniel Martinez Molina. CETSA-based evaluation of non-degraded PROTAC targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 408.