Development of isoform-selective Affimers as novel high affinity agents to study EPAC1 function

Abstract

Exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular cyclic AMP (cAMP) sensor involved in a wide variety of cellular and physiological processes. Despite an increased understanding of the different roles of EPAC1 in health and disease, reagents are lacking to study its association with cAMP nanodomains intracellularly. In this work, the non-antibody Affimer protein scaffold was used to develop isoform-selective Affimers of EPAC1. Phage-display screens against purified, biotinylated human recombinant EPAC1ΔDEP protein (149-811) identified five potential EPAC1-selective Affimers. Through dot blot and indirect ELISA assays, Affimer 780A was confirmed to be the top EPAC1-binder. Thermal shift assays revealed that 780A had a stabilising effect on the EPAC1 protein conformation in vitro and was found to not discriminate between the active and inactive conformations of EPAC1 in the presence of the EPAC1-specific agonist 007. HSQC NMR confirmed that the binding of 780A to the cyclic-nucleotide binding domain (CNBD) of EPAC1 caused a subtle conformational change. Peptide array technology and mutagenesis studies further revealed a potential interaction site for 780A in the CNBD. In EPAC1-expressing U2OS cells, 780A was shown to co-precipitate EPAC1 protein in immunoprecipitation studies. Through fluorescent and immunofluorescent confocal microscopy, 780A was able to interact with EPAC1 in COS1 cells and co-localised with both EPAC1 WT and a non-targeting EPAC1-SUMOmutant predominantly in the perinuclear and cytosolic regions, respectively. As a novel EPAC1- selective binder, 780A has the potential to be used in super resolution microscopy studies to further our understanding of the cAMP-EPAC1 signalling pathway.