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

Abstract

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.

Recent Advances of Aminopeptidases‐Responsive Small‐Molecular Probes for Bioimaging

“…In recent years, the structure of EPAP1-responsive and DPPIV-responsive probes were shown in Figure 15. [74][75][76] In 2017, Tan and his colleagues developed the first Endoplasmic Reticulum Aminopeptidase 1-responsive fluorescent probe (Probe 44). [74] After incubating the probe with Endoplasmic Reticulum Aminopeptidase 1, the fluorescent signal at 550 nm was significantly enhanced (up to 95 folds).…”

“…Given this characteristic, several small‐molecule probes have been created for the purpose of tracking Endoplasmic Reticulum Aminopeptidase 1 activity to have a better understanding of pathophysiological processes. In recent years, the structure of EPAP1‐responsive and DPPIV‐responsive probes were shown in Figure 15 [74‐76] …”

A Review on Recent Advances in Peptide‐based Fluorescence Probes and their Potential Applications

“…[1] Fluorescence probe is based on the characteristics of fluorescence, which can be used to measure metal ions, biomolecular content and label cells. [2][3][4][5][6] Fluorescent probe is usually composed of recognition group, fluorescent group and connection unit. The recognition group is mainly responsible for the recognition of objects and the fluorescent group can respond to the recognition behavior, leading the fluorescent signal changed before and after the combination with the object.…”

“…Photoluminescence refers to the natural phenomenon that objects receive light from a light source, obtain energy, and cause luminescence [1] . Fluorescence probe is based on the characteristics of fluorescence, which can be used to measure metal ions, biomolecular content and label cells [2–6] . Fluorescent probe is usually composed of recognition group, fluorescent group and connection unit.…”

Solid-phase fluorescent BODIPY–peptide synthesis via in situ dipyrrin construction

“…Fluorescent peptide/peptidomimetic-based targeting and visualization of the subcellular localization, 1,2 and probing and modulation of intra/inter-biomolecular interactions, 3,4 of the protein target of interest provides a versatile platform, either in vitro/vivo or in assays, [5][6][7] for fundamental biochemistry/ chemical biology research, [8][9][10] as well as drug discovery and development. [11][12][13][14] With the growing signicance and recent successes in peptide (and peptide-containing) therapeutics, [15][16][17][18][19][20] by virtue of their (i) scalable modular synthetic accessibility and tunability, (ii) target-specic selectivity, potency and efficacy, (iii) good biocompatibility, low systemic toxicity and easy clearance, there is a great demand for more general and exible synthetic methodologies and ligation protocols to make peptides uorescent, upon functional de novo design, for indepth biomedical investigations.…”