Visualization of membrane localization and the functional state of CB₂R pools using matched agonist and inverse...

The diversity of physiological roles of the endocannabinoid system has turned it into an attractive yet elusive therapeutic target. However, chemical probes with various functionalities could pave the way for a better understanding of the endocannabinoid system at the cellular level. Notably, inverse agonists of CB₂R - a key receptor of the endocannabinoid system - lagged behind despite the evidence regarding the therapeutic potential of its antagonism. Herein, we report a matched fluorescent probe pair based on a common chemotype to address and visualize both the active and inactive states of CB₂R, selectively. Alongside extensive cross-validation by flow cytometry, time-lapse confocal microscopy, and super-resolution microscopy, we successfully visualize the intracellular localization of CB₂R pools in live cells. The synthetic simplicity, together with the high CB₂R-selectivity and specificity of our probes, turns them into valuable tools in chemical biology and drug development that can benefit the clinical translatability of CB₂R-based drugs.