Engineering cathepsin S selective chemical probes and antibody-drug conjugates through substrate profiling with unnatural amino acids
Cysteine cathepsins, particularly cathepsin S, are important regulators of proteolytic signaling in health and disease, including cancer progression and immune modulation. Despite their therapeutic relevance, selective chemical tools to study individual cathepsins remain limited due to overlapping substrate preferences. Here we present design of cathepsin S selective chemical probes and cathepsin S-cleavable antibody-drug conjugates (ADCs) through substrate profiling with unnatural amino acids. First, Hybrid Combinatorial Substrate Library (HyCoSuL) technology incorporating a broad spectrum of unnatural amino acids was applied to comprehensively investigate the substrate specificity of cathepsin S. This approach enabled the identification of highly selective tetrapeptide motifs that served as scaffolds for the design of optimized fluorogenic substrates, irreversible inhibitors, and fluorescent activity-based probes (ABPs). These tools demonstrated high selectivity toward cathepsin S over closely related family members in both biochemical and cellular settings. We then translated these findings to develop cathepsin S-activated ADCs, incorporating the optimized peptide motifs as protease-cleavable linkers for targeted payload release. Using these linkers, we further generated MMAE-based antibody-drug conjugates directed against HER-2 and the TROP-2 proteins, demonstrating cathepsin S-dependent cytotoxicity in HER-2-positive as well as HER-2-negative/TROP-2-positive breast cancer models. Finally, we employed anti-cathepsin S antibodies in combination with mass cytometry (CyTOF) to assess the spatial distribution of cathepsin S expression in breast cancer patient samples. By correlating cathepsin S levels with tumor-associated markers such as TROP-2 and HER-2, we identified potential co-expression patterns that support the rationale for personalized therapy using antibody-drug conjugates selectively activated by cathepsin S. This integrative approach provides a comprehensive platform for profiling cathepsin S activity at the molecular, cellular, and tissue levels, with broad implications for the development of precision therapeutics and diagnostic strategies in oncology.