Identifying protease-activated targets and exploring therapeutic applications.

introductionProteases are essential enzymes that regulate protein turnover and activate signaling pathways through targeted peptide bond cleavage. While traditionally regarded as degradative agents, proteases are now recognized for their diverse roles in health and disease, particularly in cancer and viral infections. Advances in high-throughput, mass spectrometry-based technologies have enabled proteome-wide identification of protease substrates, revealing numerous potential therapeutic targets. As large-scale approaches yield expansive substrate lists, it is increasingly important to understand the roles of disease-related proteases within their specific biological contexts. AREAS COVERED: Peptide-level chemical libraries have provided more practical insights, facilitating the development of protease-targeted interventions. However, early efforts to derive inhibitors from these substrates faced challenges due to enzymatic redundancy and substrate promiscuity. Consequently, emerging research has shifted toward harnessing proteolytic activity for conditional activation of therapeutics. Since proteolytic activation can amplify therapeutic effects, protease-activated strategies, such as protease-cleavable linkers in antibody-drug conjugates, have gained interest and are now being applied to other therapeutic modalities. EXPERT OPINION: We believe that identifying substrates activated by disease-associated proteases, enabled by recent technological advances, will lead to deeper biological insights. When combined with peptide-level techniques, these discoveries can drive the development of efficient therapeutic interventions with amplified effects.