Allosteric Inhibition of Epac

Loren M Brown,Kathleen E Rogers,Nakon Aroonsakool,J Andrew Mccammon,Paul A Insel

doi:10.1074/jbc.m114.569319

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.

Highlights

exchange protein activated by cAMP (Epac) is a key mediator of cAMP signaling
Prior experimental work investigating the mechanism of Epac activation had shown that upon cAMP binding at the cyclic nucleotide-binding domain (CNBD), subsequent bending at a hinge region is an important component of protein activity [21]
Screening of Compounds That Bind to the Hinge Region of Epac—An I-TASSER generated model of Epac1 was used for Molecular Dynamics (MD) and virtual screening

Summary

Background

Epac is a key mediator of cAMP signaling. Results: We identified the hinge region of the cyclic nucleotide binding domain of Epac as a druggable region, in particular, for allosteric inhibitors. Initiation of Epac activation by cAMP occurs when it interacts with the phosphate-binding cassette of the CNBD and induces conformational changes that relieve the steric hindrance between the phosphate-binding cassette and the hinge of the CNBD, allowing the “hinge” to rotate [12, 13] This results in the release of the ionic latch due to an increase in the entropic penalty associated with preserving the auto-inhibited state and allowing the CNBD to move away from the CDC25HD [14]. Binding of cAMP induces a conformational change in which the regulatory region of Epac moves away from its catalytic region, facilitating an interaction of the cAMP; GEF, guanine nucleotide exchange factor; IFD, induced fit docking; MD, molecular dynamics; RBD, Rap1-GTP binding domain; BRET, bioluminescence resonance energy transfer; VASP, vasodilator-stimulated phosphoprotein. Subsequent studies demonstrated that a thiobarbituric acid derivative, initially identified by in silico docking at that region, prevents Epac activation in vitro and in cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION