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

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.

A G-protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these Maturation Inducing Hormones (MIHs), and understanding how they initiate the largely conserved maturation process, remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based “deorphanization” of candidate oocyte-expressed GPCRs. MIHR mutant jellyfish generated using CRISPR-Cas9 had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue 5’Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to GαS into wild type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiosis initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors including Neuropeptide Y, Gonadotropin Inhibitory Hormone, pyroglutamylated RFamide and Luqin, all upstream regulators of sexual reproduction. This identification and functional characterisation of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.

Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells

Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332 bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2′-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPβ- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPβ and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.

Xepac protein and IP3/Ca2+ pathway implication during Xenopus laevis vitellogenesis

SummaryThe objective of this study was to elucidate the signalling pathways initiated by cAMP once inside the Xenopus laevis oocyte, where it triggers and maintains vitellogenin endocytic uptake. Our results showed the presence of Xepac transcripts at all stages of oogenesis and we demonstrated that a cAMP analogue that exclusively activates Xepac, 8-CPT, was able to rescue the endocytic activity in oocytes with uncoupled gap junctions. Inhibition experiments for the IP3/Ca2+ signalling pathway showed either a complete inhibition or a significant reduction of the vitellogenic process. These results were confirmed with the rescue capability of the A-23187 ionophore in those oocyte batches in which the IP3/Ca2+ pathway was inhibited. Taking our findings into account, we propose that the cAMP molecule binds Xepac protein enabling it to activate the IP3/Ca2+ pathway, which is necessary to start and maintain X. laevis vitellogenin uptake.