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

Abstract

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 (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis and increased p62 levels. 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 hRpn13Pru-producing cancer types.

Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report

“…This group and another have previously solved the structure of Rpn13 bound to its proteasomal docking site on Rpn2 103,104 . During her talk, Walters presented unpublished data characterizing the interaction between novel small molecules and Rpn13 and the mechanism by which Rpn13 targeting leads to apoptosis 105 . The Rpn13‐binding molecules were identified by using a virtual screen based on the Rpn13:Rpn2 structure coupled with biophysical screening.…”