biostudies-literatureLu XIntramural NIH HHSU.S. Department of Health & Human Services | NIH | National Cancer Institute7318https://www.ebi.ac.uk/biostudies/studies/S-EPMC8677766biostudies-literatureUnknown12(1)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, caNature communicationsStructure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma.PMC8677766ZIA BC011490ZIA BC0116271 ZIA BC0114901 ZIA BC011627Chari RDyba MMatsuo HAndresson TSwenson REWalters KJOsei-Amponsa VChan KCLu XDas SChoudhari STran BFromont CZhao YEvans CNTarasov SGSabbasani VRTarasova NIChen XSchwieters CDKing JCfalseStructure-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, ca2021-01-01T00:00:00Z2021 Dec2026-05-09T01:27:52.315Z2022-02-11T14:48:19.637ZS-EPMC86777663491649410.1038/s41467-021-27570-4