Structure-guided design of a GSH-activated Hsp70 targeting prodrug with enhanced tumor selectivity.

The clinical application of heat shock protein 70 (Hsp70) inhibitors is limited due to their toxicity toward normal tissues. Targeting the Hsp70-Bim protein-protein interaction (PPI) represents a tumor-selective therapeutic strategy for CML. To further mitigate off-target effects, we rationally designed and synthesized three redox-responsive prodrugs (JL-15-B, JL-15-DNBS, and JL-15-S) by masking the critical phenolic hydroxyl group of Hsp70-Bim PPI inhibitor JL-15, aiming to restrict active drug release within the redox-abundant tumor microenvironment. Among these candidates, the GSH-responsive prodrug JL-15-DNBS exhibited efficient intracellular activation in TKI-sensitive and TKI-resistant CML cells. Upon activation, JL-15-DNBS restored Hsp70-Bim PPI-disrupting activity, triggered apoptosis, and downregulated oncogenic client proteins including AKT and eIF4E. Notably, JL-15-DNBS displayed a markedly improved tumor-to-normal-cell selectivity index compared with the parent JL-15. Biochemical assays indicated that the dual-activated prodrug JL-15-S failed to achieve the expected dual-triggered activation, showing only weak ROS-responsive properties, which highlights key lessons for subsequent dual-responsive prodrug structural optimization. Collectively, this work validates a tumor-microenvironment-activated prodrug strategy for Hsp70-Bim PPI inhibitors and identifies JL-15-DNBS as a promising lead compound with enhanced selectivity for CML therapy. Future investigations will focus on in vivo antitumor efficacy assessment, ADMET profiling, and further structural refinement to advance its translational potential.