Abstract

Small molecules targeting allosteric pockets of G protein–coupled receptors (GPCRs) have a great therapeutic potential for the treatment of neurologic and other chronic disorders. Here we performed virtual screening for orthosteric and putative allosteric ligands of the human dopamine D₃ receptor (D3R) using two optimized crystal-structure–based models: the receptor with an empty binding pocket (D3R^APO), and the receptor complex with dopamine (D3R^Dopa). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 μM in the D3R^APO candidate list (56% hit rate), and 8 novel ligands in the D3R^Dopa list (32% hit rate). Most ligands in the D3R^APO model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC₅₀ = 7 nM). In contrast, compounds identified by the D3R^Dopa model are predicted to occupy an allosteric site at the extracellular extension of the pocket, and they all lack the anchoring amino group. Compounds targeting the allosteric site display a variety of functional activity profiles, where behavior of at least two compounds (23 and 26) is consistent with noncompetitive allosteric modulation of dopamine signaling in the extracellular signal-regulated kinase 1 and 2 phosphorylation and β-arrestin recruitment assays. The high affinity and ligand efficiency of the chemically diverse hits identified in this study suggest utility of structure-based screening targeting allosteric sites of GPCRs.