Hydrophobic Tag Degraders Overcome Endocrine‐Resistant Breast Cancer by Recruiting HSP27‐Mediated E3 Ligase Complex for ERα Proteasomal Degradation

ABSTRACT Hydrophobic tag (HyT)‐mediated protein degradation has emerged as a pivotal tool for targeted protein degradation (TPD), yet its underlying degradation mechanism remains incompletely elucidated. Herein, we designed structurally optimized HyT‐based degraders by covalently conjugating hydrophobic amino acid tags to ERα‐targeting ligands via alkane linkers of varying lengths, identifying the lead compound VI‐10h . VI‐10h exhibited potent antiproliferative activity and efficient ERα degradation in endocrine‐resistant breast cancer cells (LCC2, MCF‐7 D538G , MCF‐7 Y537S , and MCF‐7 EGFR ) and superior antitumor activity compared to the clinical drug fulvestrant ( Ful ) in MCF‐7 and tamoxifen‐resistant LCC2 xenograft models. To elucidate the HyT‐mediated degradation mechanism, we synthesized biotin‐conjugated HyTs ( biotin‐Lys and biotin‐Trp ) and performed pull‐down assays combined with mass spectrometry. Our results unveiled that VI‐10h selectively recruits heat shock protein 27 (HSP27) as a non‐canonical E3 ligase adaptor protein, forms an ERα–HSP27–RING1 ternary complex to promote ERα degradation, disrupts estrogen‐dependent oncogenic networks, and circumvents the drug resistance associated with conventional CRBN‐ or VHL‐dependent E3 ligase‐recruiting degraders. This study clarifies a HyT‐mediated ERα degradation mechanism and supports the feasibility of using HyT degraders to overcome resistance to conventional E3 ligase‐recruiting strategies and endocrine‐resistant breast cancer, thereby establishing a molecular design strategy for next‐generation targeted degraders.