title: Isoform-specific antagonists of exchange proteins directly activated by cAMP.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Tsalkova
authors.forename: Fang C
authors.initials: FC
authors.lastname: Mei
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Chepurny
authors.forename: Colin A
authors.initials: CA
authors.lastname: Leech
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: George G
authors.initials: GG
authors.lastname: Holz
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001555:Benzene Derivatives / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D020033:Protein Isoforms / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology*; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001555:Benzene Derivatives; C000589230:ESI-05; D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; C116122:RAPGEF3 protein, human; C486707:Rapgef4 protein, mouse; D013450:Sulfones; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins