Dopamine D4 Receptor-Selective Compounds Reveal Structure-Activity Relationships that Engender Agonist Efficacy

Abstract

The dopamine D₄ receptor (D₄R) plays important roles in cognition, attention, and decision making. Novel D₄R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's disease and substance use disorders. To identify new D₄R-selective ligands, and to understand the molecular determinants of agonist efficacy at D₄R, we report a series of eighteen novel ligands based on the classical D₄R agonist A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)- N-( m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cyclic AMP inhibition assays, and molecular dynamics computational modeling. We identified several novel D₄R-selective ( K_i ≤ 4.3 nM and >100-fold vs other D₂-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor-ligand interactions that control efficacy at D₂-like receptors and may provide insights into targeted drug discovery, leading to a better understanding of the role of D₄Rs in neuropsychiatric disorders.

Figure 1

Three classic D₄R-selective partial agonists.

Scheme 1. Synthesis of 2-(4-(Pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide Analogues

Reagents and conditions: (a) triethylamine, EtOAc, RT; (b) CH₃CN, K₂CO₃, reflux, appropriate arylpiperazine or arylpiperidine.

Figure 2

Three classes of modifications to the structure of 1 resulting in differing binding and efficacy profiles at D₂-like receptors. (A) Substitution of the piperidine ring for piperazine induced a gain of efficacy at D₂R and D₃R with insubstantial changes to D₄R efficacy. (B) Substitution of the pyridine ring with a phenyl or napthyl moiety produced modest D₄R subtype selectivity improvements and lowered partial agonist efficacy at D₄R with no agonist activity at D₂R or D₃R. (C) Para-substituted pyridine rings produced highly D₄R-selective antagonists.

Figure 3

Compounds 10 (red) and 21 (gray) show similar pharmacology to parent compound 1 (black). D₄R-expressing stable cells lines were plated and compounds were assayed for agonist (A) and antagonist (B) activity on β-arrestin recruitment. Similarly, D₄R-mediated inhibition of cAMP accumulation was also examined in both agonist (C), and antagonist (D) modes, as indicated. Assays were conducted as described in the Experimental Methods; briefly, agonist assays were conducted by incubating the cells with the indicated concentration of test compound and measuring luminescence. Antagonist assays were conducted by incubating the compound with an EC₈₀ concentration of dopamine (1 μM for β-arrestin and 10 nM in cAMP) and the indicated concentration of the test compound. For cAMP assays, cells were first stimulated with 10 μM forskolin. Agonist mode assays are expressed as a percentage of the maximum dopamine response, whereas antagonist mode assays are expressed as a percentage of dopamine’s EC₈₀ response. E_max and EC₅₀ values are shown in Tables 2 and 3. Data were fit using nonlinear regression of individual experiments performed in triplicate and are shown as means ± SEM; n = 3. Dopamine and sulpiride were run during each assay as positive controls for a full agonist and full antagonist respectively (data not shown). Compounds were also tested for both agonist and antagonist activity on cells stably expressing the closely related D₂R (E) or D₃R (F). Assays were conducted as described in the Experimental Methods. Agonist mode assays (open symbols) are expressed as a percentage of the maximum dopamine response observed for each receptor, whereas antagonist mode assays (solid symbols) are expressed as a percentage of dopamine’s EC₈₀ response. E_max and EC₅₀ values are shown in Tables 2 and 3. Data were fit using nonlinear regression of individual experiments performed in triplicate and are shown as means ± SEM; n = 3.

Figure 4

Compounds 13 (yellow) and 6 (blue) show diminished agonist activity at the D₄R compared to parent compound 1 (black). D₄R-expressing stable cells lines were plated and compounds were assayed for agonist (A) and antagonist (B) activity on β-arrestin recruitment. Similarly, D₄R-mediated inhibition of cAMP accumulation was also examined in both agonist (C), and antagonist (D) modes, as indicated. Assays were conducted as described in the Experimental Methods; briefly, agonist assays were conducted by incubating the cells with the indicated concentration of test compound and measuring luminescence. Antagonist assays were conducted by incubating the compound with an EC₈₀ concentration of dopamine (1 μM for β-arrestin and 10 nM in cAMP) and the indicated concentration of test compound. For cAMP assays, cells were first stimulated with 10 μM forskolin. Agonist mode assays are expressed as a percentage of the maximum dopamine response, whereas antagonist mode assays are expressed as a percentage of dopamine’s EC₈₀ response. E_max and EC₅₀ values are shown in Tables 2 and 3. Dopamine and sulpiride were run during each assay as positive controls for a full agonist and full antagonist, respectively (data not shown). Data were fit using nonlinear regression of individual experiments performed in triplicate and are shown as means ± SEM; n = 3.

Figure 5

Compounds 12 (green) and 9 (purple) are full antagonists at the D₄R. D₄R-expressing stable cells lines were plated and compounds were assayed for agonist (A) and antagonist (B) activity on β-arrestin recruitment. Similarly, D₄R-mediated inhibition of cAMP accumulation was also examined in both agonist (C), and antagonist (D) modes, as indicated. Assays were conducted as described in the Experimental Methods; briefly, agonist assays were conducted by incubating the cells with the indicated concentration of test compound and measuring luminescence. Antagonist assays were conducted by incubating the compound with an EC₈₀ concentration of dopamine (1 μM for β-arrestin and 10 nM in cAMP) and the indicated concentration of test compound. For cAMP assays, cells were first stimulated with 10 μM forskolin. Assays were conducted as described in the Experimental Methods. Agonist mode assays are expressed as a percentage of the maximum dopamine response, whereas antagonist mode assays are expressed as a percentage of dopamine’s EC₈₀ (1 μM in β-arrestin and 10 nM in cAMP) response. E_max and EC₅₀ values are shown in Tables 2 and 3. Dopamine and sulpiride were run during each assay as positive controls for a full agonist and full antagonist respectively (data not shown). Data were fit using nonlinear regression of individual experiments performed in triplicate and are shown as means ± SEM; n = 3.

Figure 6

1 and 10 docked at D₂R. (A–D) Comparative alignment of 1 (red ligand, yellow TM domains) and 10 (blue ligand, purple TM domains) following MD simulations of the D₂R (PDB: 6CM4(26)). (E–H) Analysis of ligand interactions with specific side chains of 1 (E,G) and 10 (F,H). Although the structural difference between 1 and 10 is only a piperidine vs a piperazine ring, this drives a dramatic shift in ligand orientation in which 10 is “flipped” and rotated by 180° about its longitudinal axis, with its pyridine ring deepest in the binding pocket. This allows the basic nitrogen of the neighboring piperazine ring to engage the conserved aspartate in TM3, a common feature of dopaminergic agonists.

Figure 7

1 and 10 docked at D₃R. (A–D) Comparative alignment of 1 (red ligand, yellow TM domains) and 10 (blue ligand, purple TM domains) following MD simulations of the D₃R (PDB: 3PBL(25)). (E–H) Analysis of ligand interactions with specific side chains of 1 (E,G) and 10 (F,H). As seen in the D₂R model, 10 is also “flipped” and rotated by 180° about its longitudinal axis in the binding pocket at D₃R compared to 1. This allows for a different set of hydrophobic interactions and the engagement of the basic nitrogen of the piperazine ring to with the conserved aspartate in TM3.

Figure 8

1 and 13 docked at D₄R. (A–D) Comparative alignment of 1 (red ligand, yellow TM domains) and 13 (blue ligand, purple TM domains) following MD simulations of the D₄R (PDB: 5WIU(24)). (E–H) Analysis of ligand interactions with specific side chains of 1 (E,G) and 13 (F,H). The bulky napthyl ring of 13 shifts the overall fit within the extended binding pocket, partially disrupting the engagement of the basic nitrogen of the piperazine ring to with the conserved aspartate in TM3.

Figure 9

1 and 9 docked at D₄R. (A–D) Comparative alignment of 1 (red ligand, yellow TM domains) and 9 (blue ligand, purple TM domains) following MD simulations of the D₄R (PDB: 5WIU(24)). (E–H) Analysis of ligand interactions with specific side chains of 1 (E,G) and 9 (F,H). The inclusion of a single para substitution on the pyridine ring of 9 induces a “flipped” orientation of the ligand, in which the binding pose is rotated by 180° about its longitudinal axis, with its pyridine ring deepest in the binding pocket driving the arylamide into a deeper binding position.