PT  - JOURNAL ARTICLE
AU  - Alexey L. Chernobrovkin
AU  - Cindy Cázares-Körner
AU  - Tomas Friman
AU  - Isabel Martin Caballero
AU  - Daniele Amadio
AU  - Daniel Martinez Molina
TI  - A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts
AID  - 10.1101/2020.09.22.307926
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.09.22.307926
4099  - http://biorxiv.org/content/early/2020/09/22/2020.09.22.307926.short
4100  - http://biorxiv.org/content/early/2020/09/22/2020.09.22.307926.full
AB  - 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 to 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. IMiDs) 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).Competing Interest StatementDMM is a co-founder and shareholder of Pelago and co-inventor of patents originating from PCT/GB2012/050853. All authors are employees of Pelago Bioscience AB, Sweden. The work was carried with internal funding.