High‐throughput identification of carboxylate‐free ligands for the E3 ligase Kelch domain‐containing protein 2

Abstract Expanding the repertoire of usable E3 ubiquitin ligases remains a critical challenge in the field of targeted protein degradation. For the emerging E3 ligase Kelch domain‐containing protein 2 (KLHDC2), currently reported ligands have a carboxylate moiety in common that mimics the natural degron but compromises cellular permeability. Here we report the discovery of carboxylate‐free ligands for KLHDC2 through high‐throughput screening and cellular functional evaluation. A fluorescence polarization‐based screening assay identified NL1 as a micromolar KLHDC2 binder, which was confirmed by orthogonal biophysical methods. Although the structure–activity study did not improve affinity, a degron reporter assay demonstrated measurable cellular target engagement. Encouraged by these results, an expanded screen further identified NL2 as a neutral ligand with improved cellular activity. Computational analysis suggests that NL2 adopts a distinct binding mode, in which the conformational flexibility of two loops of KLHDC2 enables accommodation of its aromatic scaffold, while hydrophobic and π‐driven interactions compensate for the canonical ionic contacts of other carboxylate‐containing ligands. This work demonstrates that KLHDC2 can accommodate non‐carboxylate scaffolds and expands the chemical space for developing KLHDC2‐based targeted protein degradation strategies.