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.