Platform Reagents Enable Synthesis of Ligand-Directed Cova-lent 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 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.