Abstract Targeted protein degradation is a new drug discovery strategy that harnesses mainly the power of the ubiquitin-proteasome system (UPS) to degrade selectively disease-causing proteins. A key mechanism of UPS degrader molecules, including molecular glues and proteolysis-targeting chimeras (PROTACs), relies on their ability to bring target proteins into proximity with E3 ligases or complexes for ubiquitination and subsequent degradation at the proteasome. The human genome encodes at least 600 E3 ligases, of which two complexes (i.e., CUL4CRBN and CUL2VHL) are predominantly employed in the TPD field. This has raised concerns about issues like target diversity and specificity and drug resistance, and thus, prompts a strong need to look for novel, ligandable E3 ligases that can be exploited to support TPD drug discovery. To this date, we have surveyed a small panel of E3 ligase components, by utilizing the DNA-encoded library (DEL) screening technology, and identified GID4 (homolog of the yeast glucose-induced degradation deficient complex 4), substrate receptor of the CTLH E3 complex, as a top candidate that showed promising ligandability to small molecules. The GID4-binding hit molecules were validated, structure-optimized, and conjugated with a linker and BRD4-binding moiety to generate a number of PROTACs for targeting BRD4, a transcription regulator that plays an important role in promoting tumorigenesis. We demonstrated that these GID4-BRD4 PROTACs can mediate biochemical ternary binding (with EC50 1 nM) and induce BRD4 degradation in cancer cell lines (with DC50 1 uM). We also solved the crystallography structure of the ternary complex to look into potential working mechanism for one PROTAC. Using the direct-to-biology (D2B) high-throughput approach, we also have optimized the PROTACs and identified structures that can enhance degradation efficacy. Taken together, we have shown that, as a proof-of-concept, affinity-based DEL selection may help explore ligandability of proteins of interest and lead to the discovery of novel ligands and PROTACs for novel E3 ligases. Of note, our work has been corroborated by another independent study, which reported that GID4 E3 ligase can be leveraged for protein degradation (Li et al., Nat. Struct. Mol. Biol. 2025). Citation Format: Zuyuan Shen, Yunyun He, Rui Wang, Nengwei Xu, Peichuan Zhang, Lingbing Sun. Exploration of novel E3 ligase to support targeted protein degradation drug discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5164.
Shen et al. (Fri,) studied this question.