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Cells. 2019 Nov 12;8(11). pii: E1425. doi: 10.3390/cells8111425.

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

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European Screening Centre Newhouse, University of Dundee, Biocity Scotland, Bo'Ness Road, Newhouse, Lanarkshire ML1 5UH, UK.
Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Pivot Park Screening Centre, Kloosterstraat 9, 5349 AB Oss, The Netherlands.
Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh Campus, Edinburgh EH14 4AS, UK.
Institute of Chemical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK.
Department of Molecular Cancer Research, Centre of Biomedical Genetics and Cancer Genomics Centre, University Medical Centre Utrecht, 3508 TC Utrecht, The Netherlands.


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.


EPAC; Rap1; agonists; cyclic AMP; guanine nucleotide exchange factor (GEF) activity; uHTS

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