Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells

Pharmacological modulation of cannabinoid receptor type 2 (CB₂R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer’s disease. Despite the importance of CB₂R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB₂R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB₂R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB₂R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB₂R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB₂R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.