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 380,000+ 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 β-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate β-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 β-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated β-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 β-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 β-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.

Fig. 1.

MLS1547 stimulates D2R G protein–mediated signaling. (A) Structure of MLS000051547 (MLS1547). (B) Human embryonic kidney 293 cells stably expressing D2R and Gqi5 were assayed for MLS1547 stimulation of calcium accumulation, as described in Materials and Methods. Cells were stimulated with the indicated concentrations of dopamine (DA) or MLS1547 (1547). EC₅₀ and E_max values were obtained for dopamine (2.5 ± 0.3 nM and 101.7% ± 0.1%, respectively) and MLS1547 (0.37 ± 0.2 μM and 89.3% ± 4.3%, respectively) (mean ± S.E.M., n = 3). (C) CHO cells stably expressing the D2R were assayed for MLS1547 inhibition of forskolin-stimulated cAMP, as described in Materials and Methods. Cells were stimulated with the indicated concentration of dopamine or MLS1547 (1547). EC₅₀ and E_max values were obtained for dopamine (0.06 ± 0.02 μM and 100.4% ± 1.6%, respectively) and MLS1547 (0.26 ± 0.07 μM and 97.1% ± 3.7%, respectively) (mean ± S.E.M., n = 5). (D) CHO cells stably expressing D2R were assayed for sulpiride reversal of MLS1547 inhibition of forskolin-stimulated cAMP accumulation. Cells were stimulated with an EC₈₀ concentration of MLS1547 (1547) in the presence of increasing concentrations of the D2R antagonist sulpiride. The IC₅₀ value obtained for sulpiride was 22.0 ± 2.8 nM (mean ± S.E.M., n = 4). Data are representative of three to five independent experiments run in triplicate and plotted as a percentage of the maximum response observed with dopamine (B and C), or as a percentage of the response seen with an EC₈₀ concentration of MLS1547 (D), as indicated.

Fig. 2.

MLS1547 acts as an antagonist for dopamine (DA)-stimulated β-arrestin recruitment to the D2R. (A) DiscoveRx PathHunter cells were assayed for agonist-induced recruitment of β-arrestin-2 to the D2R as described in Materials and Methods. Cells were stimulated with the indicated concentrations of dopamine or MLS1547 (1547), and EC₅₀ and E_max values were obtained for dopamine (0.09 ± 0.03 μM and 99.0% ± 0.6%, respectively; mean ± S.E.M., n = 3). MLS1547 failed to stimulate measurable β-arrestin-2 recruitment. (B) Human embryonic kidney 293 cells were stably transfected with Rluc-8–fused D2R and mVenus-fused β-arrestin-2, stimulated with various concentrations of dopamine or MLS1547 (1547) as indicated, and examined for BRET as described in Materials and Methods. EC₅₀ and E_max values obtained for dopamine were 0.05 ± 0.01 μM and 99.8% ± 1.9%, respectively (mean ± S.E.M., n = 3). MLS1547 failed to stimulate any measurable D2R–β-arrestin-2 interactions. Data are representative of three to five independent experiments run in triplicate and plotted as a percentage of maximum response observed with dopamine as indicated. (C) DiscoveRx PathHunter cells were stimulated with an EC₈₀ concentration of dopamine (1 μM), then assayed for the ability of MLS1547 to antagonize this response. Data are expressed as the percentage of the maximum response observed with 1 μM dopamine and represent the mean ± S.E.M. values of three individual experiments performed in triplicate. The IC₅₀ value for MLS1547 was calculated to be 9.9 ± 0.9 μM. (D) The same cells described for the BRET assay in B were stimulated with an EC₈₀ of dopamine (1 μM) and assayed for the ability of MLS1547 to antagonize this response. Data are expressed as mean values of six independent experiments run in quadruplicate. The IC₅₀ value for MLS1547 was 3.8 ± 1.8 μM (mean ± S.E.M., n = 6).

Fig. 3.

Radioligand binding competition assay using MLS1547 and the D2R. Membranes from human embryonic kidney 293 cells stably transfected with the human D2R were harvested for radioligand competition binding assays as described in Materials and Methods. Membranes were incubated with the indicated concentrations of MLS1547 and 0.5 nM [³H]methylspiperone. The data are representative of four independent experiments and expressed as a percentage of the binding seen in the absence of any competing ligand. The K_i for MLS1547 was calculated to be 1.2 ± 0.2 μM (mean ± S.E.M., n = 4).

Fig. 4.

Pharmacophore model for G protein–biased and nonbiased agonist interactions with the D2R. (A and B) HRPR pharmacophore with aligned biased compounds MLS1547 and NCGC9141. Biased compounds align well to all four features: two aromatic (beige), one hydrophobic (green), and a positive (blue) feature. (Middle) Compared with the biased compound MLS1547, the nonbiased compound NCGC5872 (left) lacks the −Cl group and cannot align both the hydrophobic and aromatic features, whereas the different attachment points of the naphthalene ring in compounds NCGC9141 and NCGC5873 cause the former, but not the latter, to align well to the 4-point pharmacophore. In this case, one of the aromatic rings in NCGC9141 aligns the hydrophobic feature. (C and D) Docked poses of biased (cyan) and unbiased (gray) compounds in an active model of the D2R. Note the more extensive interaction of nonpolar features in the biased compounds with a hydrophobic pocket formed by residues in EL2 and TM5.