Mechanism of Action for Degraders
Targeted protein degradation is a novel modality with the potential to tackle therapeutically interesting proteins beyond the limitations of current existing approaches. In this area, CETSA® is a powerful technology which can be applied for investigating the degradation target, the mechanism of action and the efficacy of the degraders. In combination with mass spectrometry (MS), CETSA® can simultaniously evaluate compound-induced changes of the functional status of thousands of proteins and their interaction patterns in the setting of a living cell.
CETSA® Explore may therefore be used to profile protein degraders, such as molecular glues and PROTACs, to understand the mechanism of action and selectivity in intact cells. Within the same experiment, it is possible to monitor both target engagement by observing changes in protein thermal stability as well as efficacy (protein degradation) by simultaneously assessing changes in protein abundances. This makes it possible to correlate target engagement (i.e., binding to the protein target of interest and to the E3 ligase) and functional readout (i.e., compound-induced protein degradation).
In a recent study, Chernobrovkin et al. used CETSA® Explore to profile three known immuno-modulatory drugs (IMiDs), acting as molecular glues, in intact and in lysed K562 cells. In the experiments, the E3 ligase cereblon (CRBN) was confirmed as binding target for all compounds. A time-dependent decrease in abundance was observed for several known, and interestingly also for some novel, protein targets of the E3 ligase complex. In the same study, a CDK4/6 selective IMiD-based PROTAC was also profiled which confirmed its binding to CRBN as well as a time-dependent degradation of the intended target proteins CDK4 and CDK6.
Chernobrovkin AL, Cázares-Körner C, et al. A tale of two tails – efficient profiling of protein degraders by specific functional and target engagement readouts. SLAS Discov (2021) DOI: https://doi.org/10.1101/2020.09.22.307926