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Sarott_Submission_ChemRixv.pdf (1.25 MB)

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

submitted on 22.05.2020, 09:57 and posted on 28.05.2020, 15:32 by Roman Sarott, Matthias Westphal, Patrick Pfaff, Claudia Korn, David Sykes, Gazzi, Thais, Benjamin Brennecke, Kenneth Atz, Marie Weise, Yelena Mostinski, Pattarin Hompluem, koers eline, Tamara Miljuš, Nicolas 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, Teresa Grande, Samuel ruiz de martin esteban, Alexei Yeliseev, Faye Drawnel, Gabriella Widmer, Daniela Holzer, Tom van der Wel, Harpreet Mandhair, Chen-Yin Yuan, Wiliam Drobyski, Yurii Saroz, Natasha Grimsey, Michael Honer, Jürgen Fingerle, Klaus Gawrisch, Julian Romero, Cecilia J. Hillard, Zoltan V. Varga, Mario van der Stelt, Pal Pacher, Jürg Gertsch, Peter McCormick, Christoph Ullmer, Sergio Oddi, Mauro Maccarrone, Dmitry B. Veprintsev, Marc Nazare, Uwe Grether, Erick Carreira
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 context. 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 and to the detection of CB2R in human breast cancer cells.


EMC is grateful to ETH-Zürich and F. Hoffmann-La Roche for support of the research program. RCS and PP acknowledge a fellowship by the Scholarship Fund of the Swiss Chemical Industry (SSCI). MM and SO thank Dr. Lucia Scipioni and Dr. Antonio Totaro for cell culture and technical support, and Dr. Daunia Laurenti for her technical assistance in live imaging. They are also grateful to the Italian Ministry of Education, University and Research (MIUR) for partial financial support under the competitive grant PRIN 2015. JR acknowledges a grant by Ministerio de Economía y Competitividad (SAF 2016-75959-R). PJM thanks the QMUL MRC-DTP for funding for NJR. Björn Wagner, Virginie Micallef and Joelle Muller are acknowledged for the generation of PAMPA data. Some aspects of developing TR-FRET assay were supported by Swiss National Science Foundation grant 159748 to DBV.


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