Balancing Stability and Payload Release in Glutathione-Responsive PROTAC Prodrugs Targeting Prostate Cancer

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Oncology

Background:

• BET-targeting PROTACs show promise in preclinical prostate cancer models but face challenges with dose-limiting toxicities.
• Developing strategies to widen the therapeutic window for PROTACs is crucial for clinical translation.

Purpose of the Study:

• To design and evaluate a two-tier selective prodrug strategy for the BET PROTAC MZ1, enabling glutathione (GSH)-responsive and prostate cancer (PCa)-targeted delivery.
• To optimize a carbonate-disulfide prodrug motif for enhanced stability and controlled release of MZ1.

Main Methods:

• Design and synthesis of a dual-targeted prodrug integrating a PSMA ligand and a GSH-cleavable disulfide linker.
• Systematic optimization of the carbonate-disulfide motif, leading to secondary carbonate variant 2a.
• In vitro cellular studies to assess prodrug activation dependence on GSH and disulfide cleavage.
• Molecular dynamics simulations to investigate prodrug conformation and membrane permeability.

Main Results:

• The optimized secondary carbonate variant 2a demonstrated improved stability against premature hydrolysis compared to primary carbonates.
• Prodrug activation showed strong dependence on GSH, confirming controlled release of MZ1 with minimal kinetic penalty.
• Selective PSMA-mediated uptake was not demonstrated, indicating complexity in dual-targeting strategies.
• Molecular dynamics revealed intramolecular folding that may enhance passive membrane permeability.

Conclusions:

• Established design principles for stable, GSH-responsive carbonate-disulfide PROTAC prodrugs.
• Highlighted the importance of coordinated optimization of prodrug components for predictable biological behavior.
• Provided a framework for the rational development of PROTAC prodrugs with improved therapeutic windows.