Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru β-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5 -PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13 Pru ) present naturally with cell type-dependent abundance. XL5 -PROTACs preferentially target hRpn13 Pru , causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5 -PROTAC-triggered apoptosis. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13 Pru -producing cancer types. Rpn13 is a substrate receptor of the 26S proteasome and an anti-cancer drug target. Here, the authors identify and characterize XL5 , a lead compound that binds to the N-terminal Pru domain of human Rpn13 (hRpn13), solve the NMR structure of XL5 -ligated hRpn13 Pru and develop XL5 -PROTACs that preferentially target an identified hRpn13 Pru fragment present in multiple myeloma cells.