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.

Fatty acid amides (FAAs) are a family of second-messenger lipids that target cannabinoid receptors, and are known mediators of glucose-stimulated insulin secretion from pancreatic β-cells. Due to the diversity observed...

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 con-texts. Ligand-directed covalent (LDC) labeling enables the study of endogenously expressed proteins in living cells, tissues, and animals without impairment of native protein function. Herein, we employed in silico docking and molecular dynamics simulations to evaluate feasibility of LDC labeling of CB2R and guide design of LDC probes. 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 as-say. The rapid proof-of-concept verification with O-NBD probes inspired incorporation of advanced elec-trophiles suitable for experiments in live cells. To this end, novel synthetic strategies towards N-sulfonyl pyridone and N-acyl-N-alkyl sulfonamide LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular experiments. The LDC probes were characterized in vitro by a radi-oligand binding assay and TR-FRET experiments. Application of the LDC probes in flow cytometry, imag-ing flow cytometry, and confocal fluorescence microscopy confirmed specific labeling of CB2R in live cells.