Automated Profiling Of PROTAC®-Induced Cereblon Neosubstrate Degradation Using Simple Western

Many targets implicated in diseases are refractory to knockdown by traditional therapeutics like antibodies and small molecules. PROTAC® (PROteolysis TArgeting Chimeras) Degraders (hereinafter ‘Degraders’) are heterobifunctional small molecules that harness the ubiquitin-proteasome system to selectively degrade target proteins within cells. They represent an exciting new modality, repurposing small molecule chemical tools to achieve selective degradation (knockdown) of target proteins. Moreover, they have the potential to expand the ‘druggable proteome’, since they can be used to degrade proteins that, although bound, are not effectively inhibited by small molecules.

To understand the effectiveness of specific Degraders, it is important to profile Degrader neosubstrate knockdown by measuring degradation constant (DC₅₀) values, or the concentration of Degrader that induces 50% degradation of the target protein. To characterize the efficacy of Degrader molecules, researchers often run dose-response curves by way of traditional Western blotting methods. But the lengthy, manual workflow and resulting low reproducibility make it an unreliable approach for the determination DC₅₀ values. Instead, the ideal solution would be highly reproducible, allow for easy quantitation, and have a short time to results. The automated immunoassay platform known as Simple Western™ is just that, making it ideal for studying Degraders.

Here, we present data showing the power of using automated Simple Western platforms to screen panels of Degrader and IMiD compounds in order to quantify degradation activity. In this study, we demonstrate the time savings achieved by automating  these large screens as well as Simple Western’s ability to accurately quantify DC₅₀ and D_max values for specific Degraders and IMiDs.