Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1.

Elizabeth M Beck,Urszula Luchowska-Stańska,Jonathan Gillespie,Holger Rehmann,Stephen J Yarwood,Martin Timmerman,Graeme Barker,Helma Rutjes,David Morgan,Angela Cowley,John Robinson,Jolanta Wiejak,Euan Parnell,Alison Porter,Philip S Jones ,Lindsay E Robinson ,Stuart P Mcelroy ,Véronique Hamon ,Andrew Pannifer ,Angus J Morrison ,P Mahajan

doi:10.3390/cells8111425

Abstract

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

Highlights

Exchange protein activated by cyclic AMP (EPAC) proteins are emerging as drug targets with the potential to treat multiple disorders, including type 2 diabetes and cardiovascular diseases [1].EPACs are multi-domain proteins that act as a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPases, Rap1 and Rap2 [2,3]
By screening a chemically diverse library of approximately 350,000 compounds, we identified further non-cyclic nucleotide (NCN) EPAC1 agonists that are chemically distinct from I942
Dose response curves were determined with the primary EPAC1-cyclic nucleotide-binding domain (CNBD) binding assay (Table 1)

Summary

Introduction

Exchange protein activated by cyclic AMP (EPAC) proteins are emerging as drug targets with the potential to treat multiple disorders, including type 2 diabetes and cardiovascular diseases [1].EPACs are multi-domain proteins that act as a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPases, Rap and Rap2 [2,3]. EPAC1 ubiquitously expressed in most tissues, whereas EPAC2 expression is more restricted to the liver, brain, heart, and secretory organs, including the pancreas [4,5] They share the same functional domain organization and mechanisms of activation, consisting of a regulatory N-terminal region, which includes a disheveled-EGL-pleckstrin homology domain (DEP) and a cyclic nucleotide binding domain (CNBD), and a catalytic C-terminal region that includes a Ras exchange motif (REM), a Ras association domain (RA), and a CDC25 homology domain (CDC25-HD) [4,5]. We have developed an in silico docking model for interactions of the known EPAC ligand, I942 (Figure 1; [12]), with EPAC1 [13] From these studies, it is clear that EPAC2 exists as multiple conformations in dynamic equilibrium. Note that an EPAC1 structure has yet to be reported and in silico models have been constructed using an EPAC2 lysine 405 to glutamine point mutation structure, which is anticipated to be a roughly analogous docking site to that of EPAC1 [13]

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Discussion

Conclusion