Structure-Guided Discovery of Novel Dual-Site FEM1B Ligands and Assessment of Their Use in Targeted Protein Degradation

Targeted protein degradation (TPD) represents a promising approach for eliminating disease-causing proteins beyond traditional inhibition. However, the reliance on a limited number of E3 Ligases remains a major bottleneck. FEM1B, an E3 Ligase substrate receptor with multiple substrate-recognition modes, represents an attractive but underexplored TPD platform. In this study, through a structure-guided approach exploiting the spatial proximity between a druggable C-degron-binding pocket and a second binding site containing a reactive cysteine, we developed FL47, a dual-site ligand that combines extensive noncovalent interactions with targeted covalent engagement. FL47 exhibits submicromolar affinity, robust cellular target engagement, and markedly reduced cytotoxicity relative to previously reported covalent recruiters. We further applied FL47 in the development of FEM1B-based PROTACs and incorporated a chemical endocytic prodrug strategy that markedly enhanced degradation activity. This work introduces a novel dual-site binding strategy for E3 Ligase ligand discovery and broadens the potential toolbox for TPD applications.