biostudies-literatureScott DCU.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)U.S. Department of Health & Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)NIA NIH HHSHoward Hughes Medical InstituteU.S. Department of Health & Human Services | NIH | National Institute on AgingU.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer InstituteHoward Hughes Medical Institute (HHMI)Max-Planck-Gesellschaft (Max Planck Society)U.S. Department of Health & Human Services | NIH | National Cancer InstituteNCI NIH HHSU.S. Department of Health & Human Services | National Institutes of Health (NIH)U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)Max-Planck-GesellschaftNIGMS NIH HHSU.S. Department of Health & Human Services | National Institutes of Health8829https://www.ebi.ac.uk/biostudies/studies/S-EPMC11470957biostudies-literatureUnknown15(1)PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3^BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.Nature communicationsPrinciples of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2.PMC11470957R01 GM132129P30 CA0217655RO1CA247365Schulman departmentNIH P30CA021765P30 GM133893R01GM132129R01 CA247365R01 AG011085InvestigatorNIH R01AG11085Chai SCLoudon VCYang LOchoada JElledge SJRonnebaum JLee REScott DCKing MTLee HWGee CTHarper JWDharuman SLee CLi YTangallapally RJayasinghe TPaulo JASchulman BAMiller DJGriffith EChen TfalsePrinciples of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2.PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3^BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.2024-01-01T00:00:00Z2024 Oct2026-06-30T03:09:23.235Z2025-04-04T01:24:54.578ZS-EPMC114709573939604110.1038/s41467-024-52966-3