Isoform-specific antagonists of exchange proteins directly activated by cAMP

T Tsalkova, FC Mei, S Li… - Proceedings of the …, 2012 - National Acad Sciences
T Tsalkova, FC Mei, S Li, OG Chepurny, CA Leech, T Liu, GG Holz, VL Woods Jr, X Cheng
Proceedings of the National Academy of Sciences, 2012National Acad Sciences
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 …
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.
National Acad Sciences