Methods to Study the Molecular Mechanism and Drive the Design of Protein Degraders

Small-molecule degraders eliminate disease-driving proteins by hijacking the ubiquitin-proteasome system. To achieve cellular activity, protein degraders must perform a series of consecutive steps involving cell permeability, binary target engagement, and formation of a ternary complex with the target protein and a ubiquitin E3 ligase, followed by protein ubiquitination, culminating with protein degradation. Monitoring each mechanistic step of a degraders' mode of action is important to confirm its bona fide cellular activity and guide rational design and optimization. In this review, we offer an overview of how degraders work and outline the key parameters and associated methods to study each step of the mechanism. We compare and contrast biophysical and cellular in vitro assays and provide a concise framework for prioritizing and mapping them to decision stages. We also discuss the main factors affecting degrader's cellular performance and principles that have emerged to guide drug design.