Engaged but not degraded: Proteome-wide CETSA in degrader research

Targeted protein degradation has emerged as a promising strategy to address protein targets considered undruggable by small-molecule inhibitors. Moreover, proteins vulnerable to inhibition can still possess scaffolding functions that can sustain biological effects, making degradation a compelling alternative. Degraders, however, inherit the selectivity of their pharmacologically active components; for bifunctional degraders/PROTACs, two ligands contribute to this composite selectivity. Unbiased quantitative proteomics is commonly used to monitor degrader selectivity. Still, not all proteins that bind a degrader are subsequently degraded, and traditional protein-ligand interactions may persist at relevant concentrations. The Cellular Thermal Shift Assay (CETSA®) measures target engagement by detecting interactions between compounds and their cognate proteins in lysates or intact cells, without modifying compounds, proteins, or the cellular environment. Coupled to mass spectrometry (MS), CETSA enables unbiased, proteome-wide monitoring of compound-protein interactions. An MS readout can also report protein degradation when the protein of interest (POI) has a suitable half-life. Here, we demonstrate how CETSA coupled to MS can deconvolute protein binding of PROTACs targeting BRD4, CDK4/6, and CDK9, and compare the selectivity profiles of full PROTACs with their individual warheads. All investigated PROTACs and warheads showed target engagement and/or degradation of their intended targets. In intact cells, downstream biological effects were also observed; for example, effects on the CDK4/6 substrate RB1 were seen with the CDK4/6-targeting PROTAC BSJ-03-204 and its kinase-binding warhead palbociclib. In contrast, the CDK9-targeting PROTAC THAL-SNS-032 did not affect RB1, whereas its kinase-binding warhead SNS-032 did. As expected, THAL-SNS-032 induced degradation of the annotated target CDK9 and its associated cyclin T1. We also observed target engagement without degradation for additional proteins, including other kinases. GSK3A and GSK3B, as well as their downstream substrate FOXK1, were thermally shifted but not degraded by the PROTAC (consistent with the profile of SNS-032). Overall, a CETSA-MS workflow can concurrently reveal degradation, target engagement, and downstream biological effects, including liabilities arising from engagement without degradation. This integrated perspective improves selectivity assessment and supports the design and interpretation of degrader campaigns.