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.

中文翻译：

---

基于肽的荧光探针在细胞和高通量检测中成像 ERAAP 活性†

ERAAP 是一种细胞内氨基肽酶，在确定细胞通过 MHC I 类分子展示的肽库中起核心作用，ERAAP 中的功能障碍与多种疾病有关。因此，人们对开发能够在细胞中对 ERAAP 进行成像的探针产生了极大的兴趣。在本报告中，我们提出了一种称为 Ep 的荧光探针，它可以对活细胞中的 ERAAP 活动进行成像。Ep 由 10 个氨基酸的 ERAAP 底物组成，该底物具有与其偶联的供体猝灭剂对，由 BODIPY 和二硝基甲苯组成。ERAAP 裂解后 Ep 的荧光增加了 20 倍，并且能够通过荧光显微镜。此外，我们使用 Ep 开发了 ERAAP 抑制剂的高通量筛选，并筛选了包含 1460 种化合物的亲电试剂库。从这个基于 Ep 的筛选中，我们将芳香族炔酮鉴定为可以不可逆地抑制 ERAAP 活性的先导片段。鉴于 Ep 具有在细胞内成像 ERAAP 的独特能力，我们预计 Ep 将有大量应用。