Discovery and Characterization of a G Protein–Biased Agonist That Inhibits β-Arrestin Recruitment to the D2 Dopamine Receptor

Abstract

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.

Discovery of G Protein-Biased D2 Dopamine Receptor Partial Agonists

“…Therefore, we chose to use the D2R β-arrestin-2 recruitment BRET assay, where D2R arrestin recruitment is measured after only 5 minutes, and compared compound 1 to aripiprazole and the previously reported D2R G protein-biased ligand, MLS1547 ( 59 ) 27 . In HEK293T cells co-expressing D2R C-terminal-tagged renilla luciferase (Rluc), a Venus-tagged β-arrestin-2, and G protein–coupled receptor kinase 2 (GRK2), both aripiprazole and compound 59 displayed partial agonist activity for D2R-mediated β-arrestin-2 recruitment (Figure 3A, E max = 26% and 68% of quinpirole, respectively) while compound 1 exhibited no activity up to 1 µM (Figure 3A).…”

“…23–25,26 In addition, the first G protein-biased agonist of D2R was recently reported. 25,27 Biased D2R ligands are thought to stabilize different conformations of D2R leading to the preference for either G protein-dependent or G protein-independent signaling, 28–30 usually through recruitment or inhibition of β-arrestin. 31–34 Future characterization of biochemical and behavioral effects of biased D2R ligands and optimization of their drug-like properties could ultimately lead to improved antipsychotic drugs that are safer and more effective than existing antipsychotics.…”

“…While a number of β-arrestin-biased D2R agonists have been generated and progressed towards clinical development, there is only one reported G protein-biased D2R agonist. 27 …”

Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone

“…2). This residue has been implicated in the on- and off-rate kinetics and in β-arrestin biased signaling for some ligands at DRD2 and other receptors 19,20,21 . However, because of the rearrangement of EL 2 and its formation of a small helical turn (residues 182–185) in the DRD2/risperidone structure (Fig.…”

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

“…These two signaling modes harbor distinct functional properties, and in instances the same ligand can act as an agonist for one pathway but antagonist at the other. The selective or biased activation of a given pathway is commonly referred to as "functional selectivity" and can be easily demonstrated in heterologous systems especially when biased small molecule ligands are available (4). Biased GPCR ligands may have high therapeutic potential as these receptors represent the largest targets of drugs on the market.…”