Development of Cathepsin B-Responsive GalNAc-PROTACs for Hepatocyte-Targeting Protein Degradation

Targeted protein degradation (TPD) has arisen as a therapeutic revolution for eliminating disease-relevant proteins, but its tissue-specific delivery remains a critical challenge. Here, we developed an asialoglycoprotein receptor (ASGPR)-based platform for the selective degradation of target proteins in hepatocytes. By conjugating the ASGPR ligand triantennary N -acetylgalactosamine (tri-GalNAc) with a BRD4-targeted proteolysis targeting chimera (PROTAC) via a cathepsin B (CTSB)-cleavable Val-Cit-PABC linker, we generated a prototype GalNAc-PROTAC conjugate, TMU454 . TMU454 selectively degraded BRD4 in ASGPR-positive hepatocellular carcinoma cells while sparing ASGPR-negative cancer cells and normal cells. Mechanistic investigations confirmed that TMU454 -mediated BRD4 degradation is dependent on the ASGPR-mediated endocytosis, CTSB-mediated linker cleavage, and ubiquitin-proteasome system (UPS). Furthermore, a fluorescein-labeled analogue, TMU670, revealed preferential liver accumulation. Importantly, TMU454 significantly inhibited tumor growth in a Huh7-derived liver cancer xenograft model without apparent systemic toxicity. Collectively, this study establishes a versatile approach for tissue-selective protein degradation and advances targeted therapies for liver cancer.