Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):18613-8. doi: 10.1073/pnas.1210209109. Epub 2012 Oct 22.
The major physiological effects of cAMP in mammalian cells are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), as well as cyclic nucleotide-gated ion channels in certain tissues. Although a large number of PKA inhibitors are available, there are no reported EPAC-specific antagonists, despite extensive research efforts. Here we report the identification and characterization of noncyclic nucleotide EPAC antagonists that are exclusively specific for the EPAC2 isoform. These EAPC2-specific antagonists, designated as ESI-05 and ESI-07, inhibit Rap1 activation mediated by EAPC2, but not EPAC1, with high potency in vitro. Moreover, ESI-05 and ESI-07 are capable of suppressing the cAMP-mediated activation of EPAC2, but not EPAC1 and PKA, as monitored in living cells through the use of EPAC- and PKA-based FRET reporters, or by the use of Rap1-GTP pull-down assays. Deuterium exchange mass spectroscopy analysis further reveals that EPAC2-specific inhibitors exert their isoform selectivity through a unique mechanism by binding to a previously undescribed allosteric site: the interface of the two cAMP binding domains, which is not present in the EPAC1 isoform. Isoform-specific EPAC pharmacological probes are highly desired and will be valuable tools for dissecting the biological functions of EPAC proteins and their roles in various disease states.
环腺苷酸（cAMP）在哺乳动物细胞中的主要生理效应是通过两种广泛表达的细胞内 cAMP 受体，即蛋白激酶 A（PKA）和 cAMP 直接激活交换蛋白（EPAC），以及某些组织中的环核苷酸门控离子通道来传递的。尽管有大量的 PKA 抑制剂可用，但尽管进行了广泛的研究，仍没有报道 EPAC 特异性拮抗剂。在这里，我们报告了非环核苷酸 EPAC 拮抗剂的鉴定和表征，这些拮抗剂专门针对 EPAC2 同工型。这些专门针对 EPAC2 的拮抗剂，命名为 ESI-05 和 ESI-07，在体外以高亲和力抑制由 EPAC2 介导的 Rap1 激活，但不抑制 EPAC1。此外，ESI-05 和 ESI-07 能够抑制 cAMP 介导的 EPAC2 激活，但不抑制 EPAC1 和 PKA，这可以通过使用基于 EPAC 和 PKA 的 FRET 报告器或通过使用 Rap1-GTP 下拉测定在活细胞中进行监测。氘交换质谱分析进一步表明，EPAC2 特异性抑制剂通过与以前未描述的变构位点结合的独特机制发挥其同工型选择性：两个 cAMP 结合结构域的界面，在 EPAC1 同工型中不存在。同工型特异性 EPAC 药理学探针是非常需要的，并且将成为剖析 EPAC 蛋白的生物学功能及其在各种疾病状态中的作用的有价值的工具。
