A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts

Abstract

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance, and reaching “undruggable” proteins compared with traditional small-molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation; instead, one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs), understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The cellular thermal shift assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS), CETSA can simultaneously evaluate compound-induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g., immunomodulatory drugs) and PROTACs, to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment, we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e., binding to the PoI and ligases) and functional readout (i.e., degrader induced protein degradation).

Discovery of novel KRAS‒PDEδ inhibitors with potent activity in patient-derived human pancreatic tumor xenograft models

“…The CETSA assay is a useful method for the discovery of the intracellular target of inhibitors. The method evaluates the change in the stability when protein binds with the small molecules, which is fast and convenient without purifying the protein 34 , 35 . Mia Paca-2 cells were treated by compound 36l at 20 and 50 μmol/L, respectively, using compound 2 as a positive drug ( Fig.…”

Current Advances in CETSA

“…Obviously, this kind of throughput is nowhere near the CETSA HT or NanoBRET systems which can be conducted on several 384-well plates simultaneously. But these methods produce data only on the behavior of a single protein at a time, while compressed CETSA MS produces data from 7000–8000 simultaneously ( Gaetani et al, 2019 ; Chernobrovkin et al, 2021 ; Zinn et al, 2021 ; Xu Y et al, 2021 ). Thus, the compressed CETSA MS (PISA) is a powerful tool, when assessing the melting behavior of the global accessible proteome.…”

Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays

“…(2014) and could be adapted further for other protein targets. More recently, CETSA coupled with mass spectrometry (CETSA-MS) methods have been developed, which allow for simultaneous evaluation of compound-induced changes in the thermal stability of numerous proteins thus improving throughput ( Chernobrovkin et al., 2021 ). In addition, we have developed a medium throughput microtiter plate-based screening format we have named Keap1-Glow CETSA , where this assay provides a platform for the identification of small molecules targeting Keap1.…”