Anales RANF

P.12 DEVELOPMENT OF A LIVE CELL NANOBRET BINDING ASSAY FOR ADENOSINE A 2B RECEPTORS S. Hinz 1 , D. Bonasera, T. Harms, C. Vielmuth, F. Heisig, A. Behrenswerth, J. Hockemeyer, C. E. Müller University of Bonn, Bonn, Germany Adenosine receptors (ARs, subtypes A 1 , A 2A , A 2B , A 3 ) are G protein-coupled receptors which are widely distributed throughout the body and are involved in a number of physiological and pathological functions. 1 The adenosine A 2B receptor (A 2B AR) is currently in the focus of interest as a drug target in immuno-oncology, and in addition to immune cell activation, A 2B AR antagonists also display anti-proliferative and anti- angiogenic effects. Bioluminescence resonance energy transfer (BRET) assays have previously been applied to examine various aspects of receptor-ligand interactions. The novel bioluminescent donor NanoLuc luciferase, derived from Oplophorus gracilirostris luciferase, allows the use of BRET for the monitoring of ligand binding in living cells in real time. 2 In the present study we developed a novel NanoBRET-based ligand binding assay. A 2B AR antagonists (xanthine scaffold) 3,4 labeled with a fluorescent dye (a green BODIPY or a red cyanine dye) were synthesized and employed to observe BRET between the NanoLuc ® tag attached to the extracellular N-terminus of the human A 2B AR and the receptor-bound fluorescent ligand. Saturation binding experiments showed specific binding of the ligands to the A 2B AR. Kinetic studies were performed, and the calculated kinetic K D -values were in close agreement with the K D -values obtained in saturation binding experiments. The BODIPY-labeled A 2B AR antagonists showed fast association at 37°C reaching equilibrium within less than 30 min, and equilibrium binding was stable for at least 180 min. Dissociation at 37°C was induced by the addition of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Next, we performed competition binding experiments to determine the affinities of a structurally diverse set of A 2B AR ligands, agonists as well as antagonists. The obtained K i -values were consonant with the K i -values determined in radioligand binding studies. Our data showed that the novel A 2B AR NanoBRET assay will be highly useful for high- throughput screening and may replace traditional radioligand binding studies in the future. 1 Fredholm B. B. et al. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors-an update. Pharmacol Rev. 2011 , 63 , 1-34. 2 Dale C. N. et al. NanoBRET: The Bright Future of Proximity-Based Assays. Front Bioeng Biotechnol. 2019 , 7 , 1-13. 3 Jiang J. et al. A 2B Adenosine Receptor Antagonists with Picomolar Potency. J Med Chem. 2019 , 62 , 4032-4055. 4 Köse M. et al. Fluorescent-Labeled Selective Adenosine A 2B Receptor Antagonist Enables Competition Binding Assay by Flow Cytometry. J Med Chem. 2018 , 61 , 4301-4316.