CONICET | Buscador de Institutos y Recursos Humanos

G protein-coupled receptor (GPCR) kinase 5 proved to be overexpressed in failing hearts causing increased desensitization of beta-adrenergic receptors (βARs), deficit in cardiac contractility and failure progression. It has two main domains: Regulator of G protein Signaling (RGS) homology domain (RH), and protein kinase domain (KD). The mechanism of GPCRs phosphorylation by GRK5 requires the disruption of an ionic lock in RH/KD interface, leading to a more stable complex with the GPCR that enhances catalytic properties of the kinase.To obtain GRK5 inhibitors we performed a docking-based virtual screening (VS) using pdb ID 4TND to search within Enamine Advanced Collection for compounds able to bind to RH/KD interface, intending to strengthen ionic lock and avoid the catalytically competent conformation to be reached. We obtained a list of hits ordered by docking energy, ranging from -10.4 to -9.7. Using Protein Ligand Interaction Profiler tool, we evaluated non-covalent interactions between GRK5 and predicted docking poses, and observed several hydrophobic interactions and hydrogen bonds joining residues from RH and KD. Also, interesting interactions such as salt bridges, halogen bonds and π-cation were found. Accordingly, we chose 15 compounds to be purchased and evaluated in biological activity assays, for what we setted up FRET-based determinations to quantify real-time intracellular cAMP. HEKT Epac-SH187 cells co-transfected with GRK5 and β1AR or β2AR were stimulated with 10μM isoproterenol (Iso) and AUC (area under curve) values of 10min response were determined in FlexStation3 at 37°C. AUCs were reduced from 244.4±24 to 112.5±7.4 for β1AR and from 138.7±10 to 64.73±3.45 for β2AR (p