By analyzing and simulating inactive conformations of the highly homologous dopamine D₂ and D₃ receptors (D₂R and D₃R), we find that eticlopride binds D₂R in a pose very similar to that in the D₃R/eticlopride structure but incompatible with the D₂R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na⁺ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na⁺-sensitive eticlopride and Na⁺-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D₂R/risperidone structure to an extended conformation similar to that in the D₃R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D₂R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.

The dopamine D2/D3 receptor (D₂R/D₃R) agonists are used as therapeutics for Parkinson’s disease (PD) and other motor disorders. Selective targeting of D₃R over D₂R is attractive because of D₃R’s restricted tissue distribution with potentially fewer side-effects and its putative neuroprotective effect. However, the high sequence homology between the D₂R and D₃R poses a challenge in the development of D₃R selective agonists. To address the ligand selectivity, bitopic ligands were designed and synthesized previously based on a potent D₃R-preferential agonist PF592,379 as the primary pharmacophore (PP). This PP was attached to various secondary pharmacophores (SPs) using chemically different linkers. Here, we characterize some of these novel bitopic ligands at both D₃R and D₂R using BRET-based functional assays. The bitopic ligands showed varying differences in potencies and efficacies. In addition, the chirality of the PP was key to conferring improved D₃R potency, selectivity, and G protein signaling bias. In particular, compound AB04-88 exhibited significant D₃R over D₂R selectivity, and G protein bias at D₃R. This bias was consistently observed at various time-points ranging from 8 to 46 min. Together, the structure-activity relationships derived from these functional studies reveal unique pharmacology at D₃R and support further evaluation of functionally biased D₃R agonists for their therapeutic potential.