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Sarott, R.C.; Westphal, M.V.; Pfaff, P.; Korn, C.; Sykes, D.A.; Gazzi, T.; ... ; Carreira, E.M. (2020)

Development of high-specificity fluorescent probes to enable cannabinoid type 2 receptor studies in living cells

Article / Letter to editor
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....Show morePharmacological 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.Show less
All authors
Sarott, R.C.; Westphal, M.V.; Pfaff, P.; Korn, C.; Sykes, D.A.; Gazzi, T.; Brennecke, B.; Atz, K.; Weise, M.; Mostinski, Y.; Hompluem, P.; Koers, E.; Miljuš, T.; Roth, N.J.; Asmelash, H.; Vong, M.C.; Piovesan, J.; Guba, W.; Rufer, A.C.; Kusznir, E.A.; Huber, S.; Raposo, C.; Zirwes, E.A.; Osterwald, A.; Pavlovic, A.; Moes, S.; Beck, J.; Benito-Cuesta, I.; Grande, T.; Ruiz de Martin Esteban, S.; Yeliseev, A.; Drawnel, F.; Widmer, G.; Holzer, D.; Wel, T. van der; Mandhair, H.; Yuan, C.Y.; Drobyski, W.R.; Saroz, Y.; Grimsey, N.; Honer, M.; Fingerle, J.; Gawrisch, K.; Romero, J.; Hillard, C.J.; Varga, Z.V.; Stelt, M. van der; Pacher, P.; Gertsch, J.; McCormick, P.J.; Ullmer, C.; Oddi, S.; Maccarrone, M.; Veprintsev, D.B.; Nazaré, M.; Grether, U.; Carreira, E.M.
Date
2020-09-09
Journal
Journal of the American Chemical Society
Volume
142
Issue
40
Pages
16953 - 16964
DOI
doi:10.1021/jacs.0c05587
Link
https://pubs.acs.org/doi/10.1021/jacs.0c05587