title: Allosteric inhibition of Epac: computational modeling and experimental validation to identify allosteric sites and inhibitors.
abstract: 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. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), 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 ability of this compound to modulate the activity of Epac and PKA, as determined by Rap1 activity and vasodilator-stimulated phosphoprotein 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 prevents the activation of Epac1 by cAMP.
authors.affiliation: From the Departments of Pharmacology.
authors.forename: Loren M
authors.initials: LM
authors.lastname: Brown
authors.affiliation: From the Departments of Pharmacology.
authors.forename: Kathleen E
authors.initials: KE
authors.lastname: Rogers
authors.affiliation: From the Departments of Pharmacology.
authors.forename: Nakon
authors.initials: N
authors.lastname: Aroonsakool
authors.affiliation: From the Departments of Pharmacology, Chemistry and Biochemistry, and the Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California 92093.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
authors.affiliation: From the Departments of Pharmacology, Medicine and pinsel@ucsd.edu.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D000495:Allosteric Site; D000818:Animals; D002465:Cell Movement; D002470:Cell Survival; D003198:Computer Simulation; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D056004:Molecular Dynamics Simulation; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D017392:Thiobarbituric Acid Reactive Substances
chemical_list: C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; C116122:RAPGEF3 protein, human; C526973:Rapgef3 protein, rat; D017392:Thiobarbituric Acid Reactive Substances; D000242:Cyclic AMP
keywords: Allosteric Regulation; Cyclic AMP (cAMP); Drug Discovery; Epac; Guanine Nucleotide Exchange Factor (GEF); Molecular Dynamics; Virtual Screening