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

Euan Parnell,Holger Rehmann,Alison Porter,David R Adams,Stephen J Yarwood,Jolanta Wiejak,Brian O Smith,Gemma L Baillie,Stuart P Mcelroy

doi:10.1038/s41598-017-00455-7

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.

Highlights

Recent work has suggested that EPAC1 and SOCS3 may play a key role in the central control of energy balance
high throughput screening (HTS) using 8-NBD-cAMP competition assays has been limited to screens involving EPAC2, likely due to the limited fluorescence of 8-NBD-cAMP when bound to the cyclic nucleotides binding domain (CNB) of EPAC1 compared to EPAC219
In order to develop the 8-NBD-cAMP competition assay for HTS of EPAC1, the isolated cyclic nucleotide-binding domain (CNB) of EPAC1 was used, since this fragment contains the key cAMP-regulated, activation domain for EPAC1 and displays greater solubility compared to full-length recombinant EPAC127

Summary

Introduction

Recent work has suggested that EPAC1 and SOCS3 may play a key role in the central control of energy balance. It has been shown that SOCS3 may be involved in the development of insulin resistance, normally associated with T2D This it thought to occur through the disruption of insulin receptor (IR) signalling to phosphoinositide 3-kinase (PI3K) and the mitogen activated protein kinase, ERK, through its key intracellular substrate, insulin receptor substrate 1 (IRS-1)[10]. This difference may be linked to structural differences between EPAC1 and EPAC2 within the CNB that influences the selective docking of ligands to the cAMP-binding pocket[26] Both uncompetitive (CE3F4) and non-competitive EPAC1 inhibitors (5225554 and 5376753) have been identified through HTS using an in vitro EPAC1 GEF activity assay[18] and an EPAC-based bioluminescence resonance energy transfer-based assay[17], respectively. We identified a novel experimental tool to investigate the role of EPAC1 in health and disease

Objectives

Methods

Results

Conclusion