Identification of N-tert-butyloxycarbonyl protected amino acids as novel hydrophobic tags to induce targeted protein degradation

Targeted Protein Degradation (TPD) that enables the elimination of disease-causing proteins through hijacking cellular protein degradation machinery, has emerged as a transformative approach in drug discovery. Among the many available TPD strategies, such as proteolysis targeting-chimera (PROTAC), lysosome-targeting chimera (LYTAC), etc., hydrophobic tagging (HyT) has gained increasing attention for its unique mechanism to induce protein degradation by exposing hydrophobic groups on target protein surface, marking them normally smaller in size compared with Other chimeras. In this study, we explore the potential of N-Boc-protected Amino acids as hydrophobic tags, focusing on their application to degrade the Bcr-Abl fusion protein, a key driver of Chronic Myeloid Leukemia (CML). We designed and synthesized a series of degraders by conjugating N-Boc-protected Amino acids to the allosteric inhibitor GNF-2, which binds to the myristoyl pocket of Bcr-Abl. Among the 20 degraders tested, Boc-protected histidine (Boc₂His) demonstrated the most potent degradation activity. Mechanistic studies revealed that Boc₂His-induced protein degradation relies on ubiquitination of Bcr-Abl and the Proteasome pathway, with heat shock proteins HSP70 and HSP90 playing a critical role. Notably, Boc₂His-based degraders exhibited significant anti-proliferative effects in BCR-ABL-positive K562 cell line and primary CML patient cells, with minimal toxicity to non-cancerous HEK 293T cells. Furthermore, these Boc₂His-based degrader effectively degraded drug-resistant Bcr-Abl mutants, including T315I and E255K, highlighting its potential to overcome resistance to traditional tyrosine kinase inhibitors. We also extended the application of Boc₂His to Other targets, such as JAK2 and ALK, demonstrating its versatility as a degradation tag. This study underscores the promise of N-Boc-protected Amino acids, particularly Boc-protected histidine, as novel and effective hydrophobic tags for targeted protein degradation, offering a potential therapeutic avenue for CML and Other Diseases driven by dysregulated proteins.

Amino acids; Hydrophobic tagging; Targeted protein degradation.