Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

Thais Gazzi; Benjamin Brennecke; Kenneth Atz; Claudia Korn; David Sykes; Gabriel Forn-Cuni; Patrick Pfaff; Roman C. Sarott; Matthias V. Westphal; Yelena Mostinski; Leonard Mach; Malgorzata Wasinska-Kalwa; Marie Weise; Bradley L. Hoare; Tamara Miljuš; Maira Mexi; Nicolas Roth; Eline J. Koers; Wolfgang Guba; André Alker; Arne C. Rufer; Eric A. Kusznir; Sylwia Huber; Catarina Raposo; Elisabeth A. Zirwes; Anja Osterwald; Anto Pavlovic; Svenja Moes; Jennifer Beck; Matthias Nettekoven; Irene Benito-Cuesta; Teresa Grande; Faye Drawnel; Gabriella Widmer; Daniela Holzer; Tom van der Wel; Harpreet Mandhair; Michael Honer; Jürgen Fingerle; Jörg Scheffel; Johannes Broichhagen; Klaus Gawrisch; Julián Romero; Cecilia J. Hillard; Zoltan V. Varga; Mario van der Stelt; Pal Pacher; Jürg Gertsch; Christoph Ullmer; Peter J. McCormick; Sergio Oddi; Herman P. Spaink; Mauro Maccarrone; Dmitry B. Veprintsev; Erick M. Carreira; Uwe Grether; Marc Nazaré

doi:10.1039/D1SC06659E

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe†

Thais Gazzi,^a Benjamin Brennecke,^a Kenneth Atz,^b Claudia Korn,^b David Sykes,^cd Gabriel Forn-Cuni,^e Patrick Pfaff,^f Roman C. Sarott,

^f Matthias V. Westphal,^f Yelena Mostinski,^a Leonard Mach,^a Malgorzata Wasinska-Kalwa,^a Marie Weise,^a Bradley L. Hoare,^cd Tamara Miljuš,^cd Maira Mexi,^cd Nicolas Roth,^g Eline J. Koers,^cd Wolfgang Guba,^b André Alker,^b Arne C. Rufer,^b Eric A. Kusznir,^b Sylwia Huber,^b Catarina Raposo,^b Elisabeth A. Zirwes,^b Anja Osterwald,^b Anto Pavlovic,^b Svenja Moes,^b Jennifer Beck,^b Matthias Nettekoven,^b Irene Benito-Cuesta,^h Teresa Grande,^h Faye Drawnel,^b Gabriella Widmer,^b Daniela Holzer,^b Tom van der Wel,ⁱ Harpreet Mandhair,

^j Michael Honer,^b Jürgen Fingerle,^b Jörg Scheffel,^kl Johannes Broichhagen,

^a Klaus Gawrisch,^m Julián Romero,^h Cecilia J. Hillard,ⁿ Zoltan V. Varga,^mo Mario van der Stelt,ⁱ Pal Pacher,^m Jürg Gertsch,^j Christoph Ullmer,^b Peter J. McCormick,^g Sergio Oddi,^pq Herman P. Spaink,

^e Mauro Maccarrone,^qr Dmitry B. Veprintsev,^cd Erick M. Carreira,^f Uwe Grether*^b and Marc Nazaré

*^a

Author affiliations

* Corresponding authors

^a Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin, Germany
E-mail: nazare@fmp-berlin.de

^b Roche Pharma Research & Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland
E-mail: uwe.grether@roche.com

^c Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham NG7 2UH, England, UK

^d United Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, England, UK

^e Leiden University, Einsteinweg 55, 2333 CC Leiden, the Netherlands

^f Laboratorium für Organische Chemie, Eidgenössische Technische Hochschule Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland

^g William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London, London EC1M 6BQ, England, UK

^h Faculty of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain

ⁱ Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands

^j Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland

^k Dermatological Allergology, Allergie-Centrum-Charité, Department of Dermatology and Allergy, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany

^l Allergology, Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Berlin, Germany

^m National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852, USA

ⁿ Department of Pharmacology and Toxicology, Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA

^o HCEMM-SU Cardiometabolic Immunology Research Group, Department of Pharmacology and Pharmacotherapy, Semmelweis University, 1085 Budapest, Hungary

^p Faculty of Veterinary Medicine, University of Teramo, 64100 Teramo, European, Italy

^q European Center for Brain Research (CERC), Santa Lucia Foundation, 00179 Rome, Italy

^r Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy

Abstract

Despite its essential role in the (patho)physiology of several diseases, CB₂R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB₂R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB₂R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB₂R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB₂R based drugs.

This article is part of the themed collection: Most popular 2022 analytical chemistry articles

Chemical Science

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe†

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Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

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