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.cAMP-regulated guanine nucleotide exchange factor | high-throughput screening | hydrogen/deuterium exchange mass spectrometry

Spinal 5-HT7 receptors induce phrenic motor facilitation via EPAC-mTORC1 signaling

“…Aliquots of stock solutions remained viable for up to 1 wk if stored frozen (Ϫ20°C) in 100% DMSO; after this time, unused drug solutions were discarded. Prior studies confirm that EPACa is a selective EPAC activator (Christensen et al 2003;Poppe et al 2008); conversely, EPACi, PKAi, and rapamycin are regarded as selective inhibitors of EPAC (Rehmann 2013;Tsalkova et al 2012), PKA (Davies et al 2000), and mTORC1 (Davies et al 2000), respectively.…”

Role of Exchange Protein Directly Activated by Cyclic AMP Isoform 1 in Energy Homeostasis: Regulation of Leptin Expression and Secretion in White Adipose Tissue

“…As shown in Fig. 5A, suppressing Epac1 activity using an Epac-specific inhibitor, ESI-09 (13,14,20), led to dose-dependent inhibition of leptin secretion. Furthermore, consistent with the pharmacological data, knocking out Epac1 in 3T3 adipocytes using the CRISPR-Cas9 gene editing system significantly suppressed leptin secretion in differentiated 3T3-L1 adipocytes (Fig.…”

Cyclic AMP Sensor EPAC Proteins and Their Role in Cardiovascular Function and Disease

“…Novel EPAC-specific inhibitors (ESI) were identified by screening a collection of 14 400 drug-like, chemically diverse small molecules ( Figure 4). [44][45][46][47] The noncyclic-nucleotide compound named ESI-08 was the first EPAC antagonist characterized and was capable of inhibiting both EPAC1 and EPAC2 activity at 25 μmol/L in the presence of equimolar concentration of cAMP ( Figure 1). 44,45 Structure-activity relationship analysis led to the identification of >2 potent EPAC antagonists designated as HJC0197 and HJC0198 with the IC50 values of 4.0 and 5.9 μmol/L, respectively ( Figure 1).…”

“…[44][45][46][47] The noncyclic-nucleotide compound named ESI-08 was the first EPAC antagonist characterized and was capable of inhibiting both EPAC1 and EPAC2 activity at 25 μmol/L in the presence of equimolar concentration of cAMP ( Figure 1). 44,45 Structure-activity relationship analysis led to the identification of >2 potent EPAC antagonists designated as HJC0197 and HJC0198 with the IC50 values of 4.0 and 5.9 μmol/L, respectively ( Figure 1). 44 Further studies revealed that compounds ESI-05 and ESI-07 were isoform-specific antagonists exclusively inhibiting EPAC2 but not EPAC1; presumably by binding allosterically to the interface formed by the 2 CNBDs in EPAC2 (Figures 1 and 4).…”

“…44 Further studies revealed that compounds ESI-05 and ESI-07 were isoform-specific antagonists exclusively inhibiting EPAC2 but not EPAC1; presumably by binding allosterically to the interface formed by the 2 CNBDs in EPAC2 (Figures 1 and 4). 45 Another molecule named ESI-09 prevents both EPAC1 and EPAC2 GEF activity through a docking on the common CNBD-B. [46][47][48] However, ESI-09 may not act on EPAC selectively but rather behaves as nonspecific protein denaturant, especially when used at high concentration.…”