Activity-based probes that target diverse cysteine protease families

Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families. Small-molecule activity-based probes (ABPs) have recently been described as a means to track protease activities in cells, tissues and whole animals (for review see refs. 1–3). By making use of the intrinsically unique chemical reactivity of each protease class as well as the substrate-recognition domains of individual proteases, it is possible to tailor probes to react selectively either with broad classes of proteases or with individual protease targets. Although considerable progress has been made in the development of protease-specific ABPs, for several key enzyme families suitable reagents are still lacking. For example, despite successful efforts to map the substrate specificity of caspases 4,5 , most currently available ABPs for this family are limited by lack of specificity and high background labeling when applied to crude proteomes. For enzymes such as legumain, a lysosomal cysteine protease thought to be important in the initial stages of antigen presentation, only a select few inhibitors have been described and none of these has been used to generate ABPs for application in studies of legumain function 6–8 .