Discovery and Structural Characterization of Small Molecule Binders of the Human CTLH E3 Ligase Subunit GID4

[Image: see text] Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4, 16 and 67, with K(d) values of 110 and 17 μM in vitro. A parallel DNA-encoded library (DEL) screen identified five binders of GID4, the best of which, 88, had a K(d) of 5.6 μM in vitro and an EC(50) of 558 nM in cells with strong selectivity for GID4. X-ray co-structure determination revealed the basis for GID4–small molecule interactions. These results position GID4-CTLH as an E3 for TPD and provide candidate scaffolds for high-affinity moieties that bind GID4.