Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, University of Health Science Center, Houston, Texas, USA.
Sci Rep. 2017 Jul 24;7(1):6200. doi: 10.1038/s41598-017-06432-4.
Extensive functional studies of the exchange protein directly activated by cAMP (EPAC) family of signaling molecules have demonstrated that EPAC proteins play a fundamental role in several physiological and pathophysiological responses, therefore are attractive drug targets. In this report, the development of a cell-based, medium to high throughput screening assay that is capable of monitoring EPAC-mediated activation of cellular Rap1 in an isoform-specific manner is described. This assay adapts a conventional ELISA format with immobilized RalGDS-RBD as a bait to selectively capture GTP-bound active Rap1. As a result, it fills an urgent need for a cell-based EPAC assay that can be conveniently performed using microtiter plates for the discovery and/or validation of isoform-specific EPAC agonists and antagonists.
广泛的关于环磷酸腺苷（cAMP）信号分子直接激活交换蛋白（EPAC）家族的功能研究表明，EPAC 蛋白在多种生理和病理生理反应中发挥着重要作用，因此是有吸引力的药物靶点。在本报告中，描述了一种基于细胞的、中高通量筛选测定法的发展，该方法能够以同种型特异性的方式监测 EPAC 介导的细胞内 Rap1 的激活。该测定法采用了传统的 ELISA 格式，其中固定化 RalGDS-RBD 作为诱饵，选择性地捕获 GTP 结合的活性 Rap1。因此，它满足了对基于细胞的 EPAC 测定法的迫切需求，该测定法可以使用微量滴定板方便地进行，用于发现和/或验证同种型特异性的 EPAC 激动剂和拮抗剂。
