Logic-Gated Bioorthogonal In Situ Synthesis of Proteolysis-Targeting Chimeras for Precise Protein Degradation and Synergistic Immunotherapy

Targeting glutathione peroxidase 4 (GPX4) with traditional small-molecule inhibitors or conventional proteolysis-targeting chimeras (PROTACs) is limited by uncontrollable systemic toxicity. To overcome this, we engineered a nanosystem (GV@Ce6–Cu, GVCC) centered on the bioorthogonal in situ synthesis of a PROTAC, strictly governed by a tumor microenvironment (TME)-specific “AND” logic gate responsive to both overexpressed cathepsin B (CTSB) and elevated glutathione (GSH). Within the TME, CTSB specifically cleaves a peptide precursor into functional fragments, while GSH simultaneously reduces a codelivered Cu 2+ to the active Cu + catalyst. Only the concurrent action of both inputs enables the bioorthogonal ligation of the fragments in situ to form the active GPX4-degrading PROTAC. This precise synthesis orchestrates a cascade of functions. The in situ-generated PROTAC initiates ferroptosis by degrading GPX4; this effect is powerfully amplified by coreleased copper ions (driving cuproptosis) and by chlorin e6 (Ce6)-mediated photodynamic therapy (PDT), which together generate a massive reactive oxygen species burst. This cooperative induction of ferroptosis and cuproptosis, augmented by PDT, triggers robust immunogenic cell death. Consequently, GVCC treatment reprograms the immunosuppressive triple-negative breast cancer microenvironment and demonstrates potent synergy with anti-PD-L1 checkpoint blockade. This study establishes bioorthogonal in situ PROTAC synthesis as an effective precision strategy for cancer therapy.