Cy5-labeled aza-peptidyl Pro-Asn epoxide

Legumain or asparaginyl endopeptidase is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine and, occasionally, aspartic acid residues (1-3). Like other proteases, Legumain is synthesized as an inactive zymogen and is activated under acidic conditions in an autocatalytic process (4, 5). Legumain is expressed in diverse cell types and plays a key role in MHC class II–mediated antigen presentation, matrix degradation, and cysteine Cathepsin processing (6, 7). Legumain has also been shown to be implicated in various pathological conditions including parasite Infection, atherosclerosis, and tumorigenesis (1, 8-10). In mouse models of Cancer, knockdown of Legumain expression results in a marked decrease in tumor growth and metastasis, while overexpression of Legumain leads to increased tumor migration, invasion, and metastasis (1, 11). In human tumors such as carcinomas of the breast, colon, and prostate, Legumain has been shown to be overexpressed, while in normal organs like the kidney, liver, and spleen, only a limited quantity of Legumain is detectable (10, 11).

Activity-based probes (ABPs) provide a highly versatile means to monitor in vivo protease function and regulation. ABPs utilize irreversible inhibitors that can covalently modify the active site of a protease in an activity-dependent fashion (3, 12, 13). To date, most ABPs have been designed to target cysteine cathepsins and caspases, and only a few legumain-specific probes have been reported in the literature. A typical legumain-specific probe consists of a peptide scaffold, a reactive functional group, and a reporter (1, 14-18). The Cbz-Ala-Ala-Asn peptide is commonly applied as a scaffold, which is designed on the basis of the sequence of a known substrate of Legumain. Reactive functional groups, such as aza-Asn halomethylketones, aza-Asn epoxides, and aza-Asn Michael acceptors, have been used to make irreversible Legumain binding. In vitro studies have shown that these probes have a high potency against Legumain, but they also cross-react with cathepsins and caspases. The in vivo potency and specificity of these probes are largely unknown.

Investigators at Stanford University developed a class of aza-Asn epoxide ABPs with fast kinetic properties and increased selectivity for Legumain for use in in vivo imaging studies (1, 3, 16, 19). Some of the probes were also tagged with a series of cell-permeabilizing carriers. These legumain-specific probes showed a high potential for use in imaging active Legumain both in normal tissues and solid tumors. In this chapter, Legumain probe-1 (LP-1), a Cy5-labeled aza-Pro-Asn epoxide probe, is introduced (1).