Solid‐Phase Synthesis and Application of a Clickable Version of Epoxomicin for Proteasome Activity Analysis

Abstract

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be "clicked", allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.

Discovery and Development of Cyclic Peptide Proteasome Stimulators

“…Flow cytometry allows for the study of proteasomal activity in physiologically relevant conditions and presents a highthroughput alternative to traditional gel electrophoresis and western blotting techinques. [67][68][69] To test the assay, a covalent fluorescent-based probe [70,71] was applied in HEK293T cells to quantify proteasome stimulation of CyPPSs, a known proteasome stimulator (miconazole [MO]) and proteasome inhibitor (MG132) (Figure S3). The covalent fluorescent-based probe is based on the proteasome inhibitor epoxomicin, which interacts with the β5 subunit of the proteasome, with a fluorophore appended to the N-termini.…”