In addition to the ESIs identified that target both EPAC
In addition to the ESIs identified that target both EPAC1 and EPAC2, ESI-05 and ESI-07 were identified as compounds that selectively antagonise EPAC2, displaying almost no inhibition of EPAC1 at concentrations up to 100μM . Both compounds were effective inhibitors EPAC2 GEF activity towards Rap1 both in vitro and in HEK293 cells, displaying maximal inhibition between 1 and 10μM . The mechanisms of the antagonist selectivity of these compounds are ascribed to the presence of the characteristic second CNBD of EPAC2. Deuterium-exchange mass spectrometry revealed a decrease in solvent exposure on ESI-07 binding at two sites within EPAC2. The regions identified encompassed a potential binding site found at the interface between the first and second CNBDs of EPAC2. ESI-07 binding may lock EPAC2 in the closed inactive form, inhibiting both its cAMP binding and GEF functions .
Despite the apparent success of these ARCA Cy3 EGFP australia in the targeted inhibition of EPAC isoforms, doubts concerning their modes of action have been raised due to the reported denaturing properties of HJC0197 in vitro. These observations suggest that the inhibitory effects of ESI-09, ESI-08, and their derivatives are potentially nonspecific and may be linked to protein denaturation. However, docking experiments and in vivo data support a specific interaction between ESI-09 and ESI-08 with EPAC 97, 98, 99. The denaturing properties of these compounds may therefore be exacerbated by in vitro analysis or may be concentration dependent. For example, the nonspecific effects reported could be due to poor aqueous solubility of the test compounds and the fact that they were used in the study at concentrations (50–100μM) that were much higher than the effective pharmacological concentrations (<10μM) . Despite the concerns raised over ESI-08 and ESI-09, ESI-05 was confirmed to inhibit EPAC2 activity specifically without disrupting protein stability .
Recently, an EPAC1 inhibitor was identified using high-throughput screening (HTS) aimed at identifying an specific inhibitor for EPAC1 to counter the hypertrophic effects attributed to EPAC1 within the heart . The EPAC1 inhibitor CE3F4 (Table 1) was identified by directly probing GEF activity towards Rap1 in vitro1, 103, 104. Importantly, 3ECF4 was shown to act without directly disrupting the EPAC1–Rap1 interaction or cAMP binding. Although the mode of action was not disclosed, CE3F4 was observed to preferentially bind to the cAMP-bound, open conformation of EPAC1, suggesting an allosteric inhibitory mechanism . A follow-on publication described the development of the R enantiomer of CE3F4, which displays tenfold selectivity for EPAC1 over EPAC2 when compared with racemic CE3F4 . Further allosteric EPAC inhibitors have subsequently been discovered (Table 1) 84, 106.
Overall, the development of EPAC-selective antagonists has proved extremely useful for determining the biological role of EPAC in diverse biological systems. For example, the antagonist ESI-09, which inhibits EPAC2, has been shown to block myelin formation and the differentiation of Schwann cells following EPAC activation by 007 . Moreover, ESI-09 and ESI-05, which inhibit EPAC1 and EPAC2, were both found to inhibit osteoclast differentiation , whereas ESI-09, but not ESI-05, inhibits increases in cytosolic calcium in Plasmodium falciparum merozoites . EPAC-selective antagonists therefore serve as effective tool molecules that identify not only EPAC-specific effects in cells, but also which EPAC isoforms are dedicated to their control.
Concluding remarks The significance of unresolved inflammatory and immune responses in various pathologies, including T2D, rheumatoid arthritis, Crohn's disease, myeloproliferative disorders, and multiple cardiovascular diseases, is now well established. Exploiting the various inhibitory mechanisms invoked to limit these pathways therapeutically, with the aim of generating small molecules capable of either arresting or reversing disease progression, is now an important goal. Progress in understanding the role of EPAC proteins will undoubtedly help inform these approaches (Box 1).