Repurposing Tolfenamic Acid to Anchor the Uncharacterized Pocket of the PUB Domain for Proteolysis of the Atypical E3 Ligase HOIP

The E3 ligase HOIP is vital for the NF-κB pathway and is implicated in cancer and immunity. However, it remains challenging to achieve high selectivity by directly targeting the conserved catalytic RBR domain of HOIP. Herein, we identified four low-molecular-weight compounds that bind to an uncharacterized pocket of the HOIP PUB domain (HOIP^PUB). The complex structure facilitated the discovery of the first single-digit micromolar ligand of HOIP^PUB, tolfenamic acid, which exhibited over 30-fold selectivity due to the low sequence identity of the uncharacterized pocket of HOIP^PUB. Although tolfenamic acid did not block the substrate recognition and linear ubiquitination activity of HOIP, a ligand of the uncharacterized PUB pocket of HOIP (LUPH), by chemical linking pomalidomide with tolfenamic acid, degraded HOIP, reduced NEMO ubiquitination and p65 phosphorylation, and eventually inhibited NF-κB activation and breast cancer cell proliferation. Our work proposes an alternative strategy to target the nonfunctional pocket of the PUB domain with high sequence diversity to promote HOIP degradation, rather than targeting the conserved RBR domain to block the catalytic function of HOIP.