基于肽的荧光探针可在细胞内和高通量测定中对 ERAAP 活性进行成像。
A peptide-based fluorescent probe images ERAAP activity in cells and in high throughput assays.
机构信息
Department of Bioengineering, University of California at Berkeley, Berkeley, CA 94720, USA.
出版信息
Chem Commun (Camb). 2018 Jun 26;54(52):7215-7218. doi: 10.1039/c7cc09598h.
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.
ERAAP 是一种细胞内的氨肽酶，在决定 MHC Ⅰ类分子展示的肽库方面发挥着核心作用，ERAAP 的功能障碍与多种疾病有关。因此，人们非常有兴趣开发能够在细胞中成像 ERAAP 的探针。在本报告中，我们提出了一种荧光探针，称为 Ep，它可以在活细胞中成像 ERAAP 的活性。Ep 由一个 10 个氨基酸的 ERAAP 底物组成，该底物与一个供体猝灭对相连，由 BODIPY 和二硝基甲苯组成。在 ERAAP 切割后，Ep 的荧光强度增加了 20 倍，并且能够通过荧光显微镜在细胞培养物中成像 ERAAP 活性。此外，我们使用 Ep 开发了一种用于 ERAAP 抑制剂的高通量筛选方法，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，我们确定了芳基炔酮作为一种可以不可逆地抑制 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，我们预计它将有许多应用。
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