Asparaginyl endopeptidase, or
legumain, is a lysosomal
cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes.
Legumain is also up-regulated in a number of human
cancers, and recent studies suggest that it may play important functional roles in the process of
tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging
reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for
legumain. We demonstrate that optimized aza-peptidyl Asn
epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label
legumain in various normal tissues as well as in solid
tumors when applied in vivo. Interestingly, addition of
cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal
proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of
legumain function in more complex models of human disease.