A peptide-based fluorescent probe images ERAAP activity in cells and in high throughput assays

ERAAP is an intracellular amino-peptidase that plays a central role in determining the repertoire of peptides displayed by cells by MHC class I molecules, and dysfunctions in ERAAP are linked to a variety of diseases. There is therefore great interest in developing probes that can image ERAAP in cells. In this report we present a fluorescent probe, termed Ep, that can image ERAAP activity in live cells. Ep is composed of a 10 amino acid ERAAP substrate that has a donor quencher pair conjugated to it, composed of BODIPY and dinitro-toluene. Ep undergoes a 20-fold increase in fluorescence after ERAAP cleavage, and was able to image ERAAP activity in cell culture via fluorescence microscopy. In addition, we used Ep to develop a high throughput screen for ERAAP inhibitors, and screened an electrophile library containing 1460 compounds. From this Ep based screen we identified aromatic alkyne-ketone as a lead fragment that can irreversibly inhibit ERAAP activity. We anticipate numerous applications of Ep given its unique ability to image ERAAP within cells.