Development of the fluorescence polarization-based competition assay for the E3 ligase GID4

The proteolysis-targeting chimera (PROTAC) technology utilizes heterobifunctional molecules to induce targeted protein degradation through the ubiquitin-proteasome system. Structurally, PROTAC molecules consist of a target protein ligand and an E3 ligase ligand covalently linked by a suitable linker arm, forming the target protein-PROTAC-E3 ligase stable ternary complex and bringing the target protein in proximity to the E3 ligase for ubiquitination and subsequent proteasomal degradation. However, only a few E3 ligases have been used to generate effective PROTACs with limited small molecule E3 ligase ligands. Therefore, there is an urgent need to discover novel E3 ligase ligands to expand the toolbox for PROTACs. Unlike traditional E3 ligases such as ‌CRBN‌ and ‌VHL‌, GID4 E3 ligase recognizes substrates bearing N-terminal proline or other small residues through the Pro/N-degron pathway, and has already been successfully leveraged in ‌PROTAC technology. Here, we reported the development of a fluorescent probe YG11, with a K_d value of 8.1 ± 0.7 nM for GID4. With this probe, we established a robust fluorescence polarization (FP)-based competition assay for evaluation of GID4 ligands. The assay exhibited a high signal-to-noise ratio of over 20, 2.5 % DMSO tolerance, and a Z'-factor of 0.84, confirming its suitability and robustness for high-throughput screening. Thus, by enabling rapid identification of GID4 ligands, this FP competition assay promises to substantially advance PROTAC development initiatives.