Identification of a novel dopaminergic agonist that selectively activates the D 2 dopamine receptor

In order to identify novel ligands with selectivity for the D 2 dopamine (DA) receptor (DAR), we have interrogated small molecule chemical libraries using live cell functional assays and high throughput screening (HTS) techniques. Our primary HTS assay involves D 2 DAR activation of a chimeric G protein, Gqi5, which links D 2 DAR activation to Ca 2+ mobilization. This process led to the identification of a ligand (compound A) that selectively activates the D 2 DAR in comparison with other DAR subtypes. We found that compound A exhibits full agonist activity with EC 50 values ranging from 100 nM – 1 μM using three different functional assays for the D 2 DAR; Ca 2+ mobilization, inhibition of cAMP accumulation, and β‐arrestin recruitment. Using β‐arrestin recruitment assays to compare with other DARs, we found that compound A has no activity at D 1 ‐like DARs (D 1 and D 5 ), weak antagonist activity at D 4 DARs (at concentrations > 10 μM), and either weak partial agonist (<20% of the DA response) or full antagonist activity at D 3 DARs. Radioligand binding assays revealed that compound A exhibits Ki values of ~1 μM and ~100 nM for the D 2 and D 3 DARs, respectively. In summary, compound A is a full and selective agonist at the D 2 DAR, although it is also functions as a D 3 DAR antagonist. This is the first known compound that functionally discriminates between the D 2 and D 3 DARs and selectively activates the D 2 DAR in the absence of D 3 DAR activation.