In-Cell Approach to Evaluate E3 Ligases for Use in Targeted Protein Degradation

A major challenge in evaluating the suitability of ∼700 known and putative E3 ligases for target protein degradation (TPD) is the lack of ligase-specific binders. Here, we use genetic code expansion (GCE) to express in living cells an E3 ligase with a site-specifically encoded, tetrazine-containing noncanonical amino acid (Tet-ncAA). Then, using click chemistry, we conjugate the incorporated Tet with a strained trans -cyclooctene (sTCO) tethered to a neosubstrate protein binder. The resulting covalent E3 ligase–binder construct can then be evaluated for the TPD of the neosubstrate. We first demonstrate that cereblon (CRBN) has a rather high plasticity for TPD by studying CRBN containing Tet-ncAA at a variety of surface positions. When these CRBN forms are covalently tethered to an sTCO-linker-JQ1 reagent, they all successfully recruit BRD2/4 for degradation, with the efficiency depending on the placement of the Tet-ncAA and the linker length. The results highlight the ability of this approach to map E3 surfaces and identify optimal TPD interfaces and pockets. Applying this strategy to speckle-type POZ protein (SPOP), an E3 ligase with no known specific ligand, we demonstrate that multiple sites on its surface can support TPD, revealing the potential for PROTAC-type development. This E3-ligand-free degrader (ELF degrader) platform preserves the native state of E3 ligases, enables the interrogation of any E3 surface region in live cells, and is applicable to a broad range of E3 ligases. ELF degraders represent a versatile approach to define functional degron sites, guide degrader design, and unlock new E3 ligases, those without known ligands, for therapeutic applications.