Structure-based ligand discovery targeting orthosteric and allosteric pockets of dopamine receptors

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 (D3RDopa). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 ?M in the D3RAPO candidate list (56 hit rate), and 8 novel ligands in the D3RDopa list (32 hit rate). Most ligands in the D3RAPO model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC 50 57 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 ?-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.