A high-throughput screening campaign was conducted to interrogate a 380,0001 small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and b-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate b-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit b-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopaminestimulated b-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate b-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate b-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

The D 1 dopamine receptor (D 1 R) has been proposed to form a hetero-oligomer with the D 2 dopamine receptor (D 2 R), which in turn results in a complex that couples to phospholipase Cmediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose-response curves assaying intracellular calcium mobilization in cells heterologously expressing the D 1 and D 2 subtypes, either alone or in combination, and using subtype selective ligands revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D 1 -D 2 heteromer-selective ligand, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol (SKF83959), found no stimulation of calcium release, but it did find a broad range of cross-reactivity with other G protein-coupled receptors. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Overexpression of G qa with the D 1 and D 2 dopamine receptors enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing G qa with only the D 1 R. Inactivation of G i or G s with pertussis or cholera toxin, respectively, largely, but not entirely, reduced the calcium response in D 1 R and D 2 R cotransfected cells. Moreover, sequestration of G bg subunits through overexpression of G protein receptor kinase 2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggest that the mechanism of D 1 R/D 2 R-mediated calcium signaling involves more than receptor-mediated G q protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition, SKF83959 may not exhibit selective activation of D 1 -D 2 heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.