Discovering Uncharted Binding Pockets on E3 Ligases Leads to the Identification of FBW7 Allosteric Modulators

E3 ligases are key regulators of the ubiquitin‐proteasome system (UPS) and have emerged as attractive drug target candidates for precise therapeutic intervention. Additionally, their ligands are extremely valuable as handles for Targeted Protein Degradation (TPD). However, only a limited number of E3 ligases have been targeted with small molecules. An efficient approach to identify ligandable surfaces on 22 structurally diverse E3 ligases has been developed, revealing that they offer significant binding opportunities through allosteric pockets. As a proof of concept, an allosteric pocket identified in FBW7 has been targeted, leading to the discovery of the first potent and reversible small‐molecule binders of this E3 ligase. Biophysical and structural studies have confirmed the binding site, while functional cell assays have showed that some of these molecules act as allosteric enhancers of c‐MYC and c‐JUN degradation in an FBW7‐dependent manner. These allosteric modulators of E3 ligases represent a novel mechanism of action in the TPD landscape and could be used as PROTAC handles.