Modular Development of Enzyme-Activatable Proteolysis Targeting Chimeras for Selective Protein Degradation and Cancer Targeting.

As an emerging therapeutic modality, proteolysis targeting chimeras (PROTACs) indiscriminately degrade proteins in both healthy and diseased cells, posing a risk of on-target off-site toxicity in normal tissues. Herein, we present the modular development of enzyme-activatable PROTACs, which utilize enzyme-recognition moieties to block protein degradation activities and can be specifically activated by elevated enzymes in cancer cells to enable cell-selective protein degradation and cancer targeting. We identified the methylene alkoxy carbamate (MAC) unit as an optimal self-immolative linker, possessing high stability and release efficiency for conjugating enzyme-recognition moieties with PROTACs. Leveraging the MAC linker, we developed a series of enzyme-activatable PROTACs, harnessing distinct enzymes for cancer-cell-selective protein degradation. Significantly, we introduced the first dual-enzyme-activatable PROTAC that requires the presence of two cancer-associated enzymes for activation, demonstrating highly selective protein degradation in cancer cells over nonmalignant cells, potent in vivo antitumor efficacy, and no off-tumor toxicity to normal tissues. The broad applicability of enzyme-activatable PROTACs was further demonstrated by caging other PROTACs via the MAC linker to target different proteins and E3 ligases. Our work underscores the substantial potential of enzyme-activatable PROTACs in overcoming the off-site toxicity associated with conventional PROTACs and offers new opportunities for targeted cancer treatment.