Protease‐activated prodrugs: strategies, challenges, and future directions

Proteases play critical roles in virtually all biological processes, including proliferation, cell death and survival, protein turnover, and migration. However, when dysregulated, these enzymes contribute to the progression of multiple diseases, with cancer, neurodegenerative disorders, inflammation, and blood disorders being the most prominent examples. For a long time, disease-associated proteases have been used for the activation of various prodrugs due to their well-characterized catalytic activity and ability to selectively cleave only those substrates that strictly correspond with their active site architecture. To date, versatile peptide sequences that are cleaved by proteases in a site-specific manner have been utilized as bioactive linkers for the targeted delivery of multiple types of cargo, including fluorescent dyes, photosensitizers, cytotoxic drugs, antibiotics, and pro-antibodies. This platform is highly adaptive, as multiple protease-labile conjugates have already been developed, some of which are currently in clinical use for cancer treatment. In this review, recent advancements in the development of novel protease-cleavable linkers for selective drug delivery are described. Moreover, the current limitations regarding the selectivity of linkers are discussed, and the future perspectives that rely on the application of unnatural amino acids for the development of highly selective peptide linkers are also presented.