期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2012
卷号：109
期号：45
页码：18613-18618
DOI：10.1073/pnas.1210209109
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
