title: A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43-Ezrin Interaction Disruptors Using IncuCyte ZOOM.
abstract: Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.
authors.affiliation: 1 Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway.|2 Biotechnology Centre, University of Oslo, Oslo, Norway.
authors.forename: Aleksandra R
authors.initials: AR
authors.lastname: Dukic
authors.affiliation: 2 Biotechnology Centre, University of Oslo, Oslo, Norway.
authors.forename: David W
authors.initials: DW
authors.lastname: McClymont
authors.affiliation: 1 Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway.|2 Biotechnology Centre, University of Oslo, Oslo, Norway.|3 K.G. Jebsen Centre for Cancer Immunotherapy, University of Oslo, Oslo, Norway.|4 Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway.
authors.forename: Kjetil
authors.initials: K
authors.lastname: Taskén
mesh_terms: D000818:Animals; D002450:Cell Communication / Q000187:drug effects; D018031:Connexin 43 / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D021621:Imaging, Three-Dimensional; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000494:pharmacology; D051381:Rats; D000099149:Ezrin
chemical_list: D018031:Connexin 43; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003598:Cytoskeletal Proteins; D005452:Fluoresceins; D011506:Proteins; D000099149:Ezrin; C400224:Ht 31 protein, synthetic; C007740:fluorexon
keywords: Cx43; Ezrin; gap junction communication; high throughput; protein-protein interaction; screening assay