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

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

The future of EPAC-targeted therapies: agonism versus antagonism

yesPharmaceutical manipulation of cAMP levels exerts beneficial effects through the regulation of the exchange protein activated by cAMP (EPAC) and protein kinase A (PKA) signalling routes. Recent attention has turned to the specific regulation of EPAC isoforms (EPAC1 and EPAC2) as a more targeted approach to cAMP-based therapies. For example, EPAC2-selective agonists could promote insulin secretion from pancreatic β cells, whereas EPAC1-selective agonists may be useful in the treatment of vascular inflammation. By contrast, EPAC1 and EPAC2 antagonists could both be useful in the treatment of heart failure. Here we discuss whether the best way forward is to design EPAC-selective agonists or antagonists and the current strategies being used to develop isoform-selective, small-molecule regulators of EPAC1 and EPAC2 activity

Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins