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

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 Kd 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 Kd of 5.6 μM in vitro and an EC50 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.