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Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1
Elizabeth M Beck1, Euan Parnell2, Angela Cowley1
1European Screening Centre Newhouse, University of Dundee, Biocity Scotland, Bo'Ness Road, Newhouse, Lanarkshire ML1 5UH, UK.
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.
Insights
Researchers identified SY009, a novel non-cyclic nucleotide activator, that selectively targets EPAC1 (Exchange protein directly activated by cAMP 1). This discovery offers potential therapeutic strategies for diabetes and cardiovascular diseases by modulating Rap GTPase signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cyclic AMP (cAMP) activates EPAC1 and EPAC2, which regulate Rap GTPases controlling crucial cellular functions.
- EPAC1 and EPAC2 activation holds therapeutic potential for diabetes and cardiovascular diseases.
- Developing synthetic agonists for EPAC1 and EPAC2 is a key research objective.
Purpose of the Study:
- To identify novel EPAC agonists using a high-throughput screening assay.
- To characterize the potency and selectivity of identified EPAC1 binders.
- To validate novel compounds as EPAC1 activators in cellular assays.
Main Methods:
- Ultra-high-throughput screening (uHTS) assay utilizing a fluorescent cAMP analogue (8-NBD-cAMP) to detect EPAC1 CNBD binding.
- Synthesis and testing of a library of SY000 analogues, including SY009.
- Orthogonal assays such as competition binding, microscale thermophoresis, in vitro activity assays, and Rap1 GTPase activation assays.
Main Results:
- A benzofuran oxaloacetic acid derivative, SY000, was identified as a moderate EPAC1 binder.
- SY009, an analogue of SY000, demonstrated a 10-fold increase in EPAC1 binding potency.
- SY009 confirmed as an EPAC1 agonist in vitro and selectively activated EPAC1 over EPAC2 in cellular assays.
Conclusions:
- SY009 represents a new class of non-cyclic nucleotide (NCN) EPAC1 activators.
- The selective activation of EPAC1 by SY009 provides a promising avenue for therapeutic development.
- Further research into NCN EPAC1 activators could lead to novel treatments for cAMP-mediated diseases.
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