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.

Highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231.

Highly selective fluorescent activity-based probe for the visualization of cathepsin B in cancer cells.

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes—ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.