Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells
Roman C. Sarott, Matthias V. Westphal, Patrick Pfaff, Claudia Korn, David A. Sykes, Thais Gazzi, Benjamin Brennecke, Kenneth Atz, Marie Weise, Yelena Mostinski, Pattarin Hompluem, Eline J. Koers, Tamara Miljuš, Nicolas J. Roth, Hermon Asmelash, Man Vong, Jacopo Piovesan, Wolfgang Guba, Arne C. Rufer, Eric A. Kusznir, Sylwia Huber, Catarina Raposo, Elisabeth A. Zirwes, Anja Osterwald, Anto Pavlovic, Svenja Moes, Jennifer Beck, Irene Benito‐Cuesta, María T. Grande, Samuel Ruiz de Martı́n Esteban, Alexei Yeliseev, Faye Drawnel, Gabriella Widmer, Daniela Holzer, Tom van der Wel, Harpreet Mandhair, Cheng-Yin Yuan, William R. Drobyski, Yurii Saroz, Natasha L. Grimsey, Michael Honer, Jürgen Fingerle, Klaus Gawrisch, Julián Romero, Cecilia J. Hillard, Zoltán V. Varga, Mario van der Stelt, Pál Pacher, Jürg Gertsch, Peter J. McCormick, Christoph Ullmer, Sergio Oddi, Mauro Maccarrone, Dmitry B. Veprintsev, Marc Nazaré, Uwe Grether, Erick M. Carreira
Abstract
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer’s disease.