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

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.