Allosteric Inhibition of Epac: Computational Modeling and Experimental Validation to Identify Allosteric Sites and Inhibitors [Signal Transduction]

September 2nd, 2014 by Brown, L. M., Rogers, K. E., Aroonsakool, N., McCammon, J. A., Insel, P. A.

Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. We found that the conserved hinge region of the cyclic nucleotide binding domain (CNBD) of Epac1 is a potentially druggable region of the protein. Using a BRET-based assay (CAMYEL), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the compound′s ability to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that binds to the hinge region of the CNBD of Epac1 and Epac2 to prevent their activation by cAMP.
  • Posted in Journal of Biological Chemistry, Publications
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