Single-position ligand modifications tune CB2R activity by targeting the toggle switch

: Cannabinoid receptor type 2 (CB2R) is a prominent class A G protein-coupled receptor (GPCR) and is a therapeutic target of interest for inflammatory diseases, pain management, and neurodegenerative disorders. We report the development of ligands based on HU-308 that share a single central scaffold but bear diverse sidechains, enabling controlled modulation of GPCR activation. Structural modifications at a single position of the parent ligand allow modulation of the single-residue toggle switch of CB2R, Trp2586.48, and thereby control over receptor activity. A continuum of functional outcomes is achieved through interaction of the ligands with the CB2R toggle switch, leading to full agonism, partial agonism, neutral antagonism, or partial inverse agonism. Several low-efficacy ligands display protean behavior across assays, underscoring context-dependent modulation of CB2R and its importance in profiling such ligands. A notable compound within this series is CF3-substituted (S)-1, which displays distinct CB2R affinity, potency, and a biased CB2R signaling profile. We provide a rationale based on molecular dynamics simulations for the unique pharmacological profile observed and suggest that stabilization of an active receptor conformation occurs by close-contact interaction of (S)-1 with the CB2R toggle switch. Our findings demonstrate that strategic structural modifications of class A GPCR ligands may, by targeting a receptor's toggle switch, shift ligands to different positions along the efficacy spectrum, independent of their parent scaffold's original functional profile.