Identification of a Novel, Small Molecule Partial Agonist for the Cyclic AMP Sensor, EPAC1

Abstract

Screening of a carefully selected library of 5,195 small molecules identified 34 hit compounds that interact with the regulatory cyclic nucleotide-binding domain (CNB) of the cAMP sensor, EPAC1. Two of these hits (I942 and I178) were selected for their robust and reproducible inhibitory effects within the primary screening assay. Follow-up characterisation by ligand observed nuclear magnetic resonance (NMR) revealed direct interaction of I942 and I178 with EPAC1 and EPAC2-CNBs in vitro. Moreover, in vitro guanine nucleotide exchange factor (GEF) assays revealed that I942 and, to a lesser extent, I178 had partial agonist properties towards EPAC1, leading to activation of EPAC1, in the absence of cAMP, and inhibition of GEF activity in the presence of cAMP. In contrast, there was very little agonist action of I942 towards EPAC2 or protein kinase A (PKA). To our knowledge, this is the first observation of non-cyclic-nucleotide small molecules with agonist properties towards EPAC1. Furthermore, the isoform selective agonist nature of these compounds highlights the potential for the development of small molecule tools that selectively up-regulate EPAC1 activity.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Subsequently, noncompetitive EPAC1 inhibitors (compounds 5225554 and 5376753) have also been identified through a virtual compound screening of a library from the Chembridge database against the apo-EPAC structural model (111,112). More recently, Parnell et al (809) applied the 8-NBD-cAMP displacement HTS assay to isolated CNB domains of EPAC1 or EPAC2 and identified additional compounds I178, I288, and I942 that could modestly suppress cAMP-mediated EPAC1 and EPAC2 activation. Interestingly, when tested in the absence of cAMP, I942 was able to weakly activate EPAC1 with apparent AC 50 values of 40 M and a maximal activity around 10% compared with cAMP, suggesting that I942 may act as a partial agonist for EPAC1 (809).…”

“…More recently, Parnell et al (809) applied the 8-NBD-cAMP displacement HTS assay to isolated CNB domains of EPAC1 or EPAC2 and identified additional compounds I178, I288, and I942 that could modestly suppress cAMP-mediated EPAC1 and EPAC2 activation. Interestingly, when tested in the absence of cAMP, I942 was able to weakly activate EPAC1 with apparent AC 50 values of 40 M and a maximal activity around 10% compared with cAMP, suggesting that I942 may act as a partial agonist for EPAC1 (809). Additional pharmacological characterization and medicinal chemistry optimization are necessary to improve the potency and utility of these compounds.…”

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology

“…The fluorescent cAMP analogue 8-NBD-cAMP was used as a ligand of the isolated CNBDs of Epac1 and 2 to screen for binding competitors [30]. Compounds, named I942 and I178, were found to inhibit 8-NBD-cAMP binding, suggesting interaction with Epac CNBDs.…”

“…Compounds, named I942 and I178, were found to inhibit 8-NBD-cAMP binding, suggesting interaction with Epac CNBDs. In a functional fluorescence-based Rap1 GEF assay, I942 compound (Table 1) showed partial agonist activity towards Epac1 but not Epac2 or PKA, with an EC50 = 50 µM and a maximal potency of less than 10% of that of cAMP [30]. I942 was reported to promote Epac1-Rap activation in HEK293T cells stably expressing Epac1, and to induce SOCS3 expression and suppress Epac1-dependant IL6-stimulated JAK/STAT3 signalling in cultured vascular endothelial cells [31].…”

Structure-activity relationships of 2-substituted phenyl- N -phenyl-2-oxoacetohydrazonoyl cyanides as novel antagonists of exchange proteins directly activated by cAMP (EPACs)

“…15 Most EPAC agonists are derivatives of cAMP, 16 while non-cyclic nucleotide ligands usually display EPAC inhibitory activities, except a very recently reported small molecule partial agonist with modest potency. 17 Cheng, et al developed a sensitive and robust fluorescence-based high throughput (HTS) assay, 18 which led to the discovery of a series of non-cyclic nucleotide EPAC inhibitors. 19 After extensive modifications by our team, dihydropyrimidine 2 (HJC0198, Fig.…”