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Drug Derived Fluorescent Probes for the Specific Visualization of Cannabinoid Type 2 Receptor - A Toolbox Approach
preprintsubmitted on 12.11.2019, 08:54 and posted on 22.11.2019, 01:43 by Gazzi, Thais, Benjamin Brennecke, Kenneth Atz, Claudia Korn, David Sykes, Roman C. Sarott, Matthias V. Westphal, Patrick Pfaff, Marie Weise, Yelena Mostinski, Bradley L. Hoare, Tamara Miljuš, Maira Mexi, Wolfgang Guba, André Anker, Arne C. Rufer, Eric A. Kusznir, Sylwia Huber, Catarina Raposo, Elisabeth A. Zirwes, Anja Osterwald, Anto Pavlovic, Svenja Moes, Jennifer Beck, Irene Benito-Cuesta, Teresa Grande, Faye Drawnel, Gabriella Widmer, Daniela Holzer, Tom van der Wel, Harpreet Mandhair, Yurii Saroz, Natasha Grimsey, Michael Honer, Jürgen Fingerle, Klaus Gawrisch, Julian Romero, Cecilia J. Hillard, Peter J. McCormick, Zoltan V. Varga, Mario van der Stelt, Pal Pacher, Jürg Gertsch, Christoph Ullmer, Sergio Oddi, Mauro Maccarrone, Dmitry B. Veprintsev, Erick M. Carreira, Uwe Grether, Marc Nazare
Cannabinoid type 2 receptor (CB2R) is a fundamental part of the endocannabinoid signaling system (eCB system), and is known to play an important role in tissue injury, inflammation, cancer and pain. In stark contrast to its significance, the underlying signaling mechanisms and tissue expression profiles are poorly understood. Due to its low expression in healthy tissue and lack of reliable chemical tools, CB2R visualization in live cells remains uncharted. Here we report the development of a drug derived toolbox of highly potent, CB2R-selective fluorescent probes based on reverse design. Extensive validation in several applications such as CB2R detection in flow cytometry and time-resolved imaging, and the development of a novel fluorescent-based TR-FRET assay to generate kinetic and equilibrium binding data demonstrate the high versatility of our toolbox. These probes are the first to preserve affinity and efficacy in both human and mouse CB2R, a crucial aspect for preclinical translatability, and to enable imaging of CB2R internalization in living cells using confocal microscopy.