title: Development of near-infrared fluorophore (NIRF)-labeled activity-based probes for in vivo imaging of legumain.
abstract: 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.
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305-5324, USA.
authors.forename: Jiyoun
authors.initials: J
authors.lastname: Lee
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000032:analysis*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D004852:Epoxy Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000201:enzymology*; D019265:Spectroscopy, Near-Infrared; D023041:Xenograft Model Antitumor Assays; D000099240:Asparaginyl Endopeptidase
chemical_list: D001216:Asparagine; D002232:Carbocyanines; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D004852:Epoxy Compounds; D005456:Fluorescent Dyes; D000099240:Asparaginyl Endopeptidase; C085321:cyanine dye 5; C549218:legumain probe-1