Investigation of the binding and functional properties of extended length D3 dopamine receptor-selective antagonists

Abstract

The D3 dopamine receptor represents an important target in drug addiction in that reducing receptor activity may attenuate the self-administration of drugs and/or disrupt drug or cue-induced relapse. Medicinal chemistry efforts have led to the development of D3 preferring antagonists and partial agonists that are >100-fold selective vs. the closely related D2 receptor, as best exemplified by extended-length 4-phenylpiperazine derivatives. Based on the D3 receptor crystal structure, these molecules are known to dock to two sites on the receptor where the 4-phenylpiperazine moiety binds to the orthosteric site and an extended aryl amide moiety docks to a secondary binding pocket. The bivalent nature of the receptor binding of these compounds is believed to contribute to their D3 selectivity. In this study, we examined if such compounds might also be "bitopic" such that their aryl amide moieties act as allosteric modulators to further enhance the affinities of the full-length molecules for the receptor. First, we deconstructed several extended-length D3-selective ligands into fragments, termed "synthons", representing either orthosteric or secondary aryl amide pharmacophores and investigated their effects on D3 receptor binding and function. The orthosteric synthons were found to inhibit radioligand binding and to antagonize dopamine activation of the D3 receptor, albeit with lower affinities than the full-length compounds. Notably, the aryl amide-based synthons had no effect on the affinities or potencies of the orthosteric synthons, nor did they have any effect on receptor activation by dopamine. Additionally, pharmacological investigation of the full-length D3-selective antagonists revealed that these compounds interacted with the D3 receptor in a purely competitive manner. Our data further support that the 4-phenylpiperazine D3-selective antagonists are bivalent and that their enhanced affinity for the D3 receptor is due to binding at both the orthosteric site as well as a secondary binding pocket. Importantly, however, their interactions at the secondary site do not allosterically modulate their binding to the orthosteric site.

Keywords: Allosteric; Bitopic; Bivalent; D(3) receptor; Dopamine antagonists; Dopamine receptor.

Fig. 1

Structures of extended-length D₃ receptor-selective ligands (A) and derived synthons (B) used in this study.

Fig. 2. Effect of secondary synthons on the ability of primary orthosteric synthons to compete for D₃ receptor binding

Radioligand competition binding assays were performed as described in the Experimental Procedures. Competition isotherms of primary synthons (A) BAK 3-33; (B) BAK 3-44; (C) BAK 2-46; (D) BAK 3-48 or (E) BAK 3-51 in the absence (●) or presence of 50 μM of secondary synthons BAK 2-50, BAK 2-51, BAK 3-21, or NMP 8-073, as indicated. Data in (A–E) are plotted as mean±SEM, n=3, performed in triplicate. Results are expressed as a percentage of specific [³H]methylspiperone binding in the absence of any competitor (% control). K_i values were calculated from observed IC₅₀ values from individual experiments using the Cheng–Prusoff equation and are displayed in Tables 1 and 2. (F) Competition isotherm of BAK 2-50 conducted using [³H]methylspiperone and the indicated concentrations of BAK 2-50. Results are representative of two independent experiments performed in triplicate.

Fig. 3. The effect of a secondary synthon on the inhibition of dopamine-stimulated β-arrestin recruitment by orthosteric synthons

β-arrestin recruitment assays were performed as described in the Experimental Procedures. Cells were stimulated with an EC₉₀ concentration of dopamine (2 μM) and the indicated concentrations of test compound. Data are plotted as a percentage of the response seen with 2 μM dopamine in the absence of compound as mean±SEM, n=3. (A) Inhibitory concentration response curves of BAK 2-46 or sulpiride alone or in the presence of 50 μM BAK 2-50, as indicated. IC₅₀ (95% CI) values obtained from mean graphs of BAK 2-46 and BAK 2-46+BAK 2-50 were 9.7 μM (3.5–27) and 4.3 μM (2.1–8.7) respectively, and from mean graphs of sulpiride and sulpiride+BAK 2-50 were 0.5 μM (0.4–0.7) and 0.5 μM (0.3–1) respectively. (B) Inhibitory concentration response curves of BAK 3-44 alone or in the presence of 50 μM BAK 2-50, as indicated. Sulpiride data is re-plotted here from panel A for data comparison. IC₅₀ (95% CI) values obtained from mean graphs of BAK 3-44 and BAK 3-44+BAK 2-50 were 6.0 μM (2.4–15) and 11.0 μM (3.5–35), respectively. No significant difference between IC₅₀ values of each compound upon the addition of BAK 2-50 was observed (Student’s t-test (p<0.05)).

Fig. 4. The effect of secondary synthons on dopamine-stimulated β-arrestin recruitment to the D₃ receptor

β-arrestin recruitment assays were performed as described in Section 2. Cells were stimulated with the indicated concentrations of dopamine either alone or presence of 50 μM BAK 2-50, BAK 2-51, BAK 3-21, or NMP 8-073, as indicated. Data are representative of three independent experiments run in triplicate and plotted as average relative luminescence units (RLU)±SEM. Dopamine EC₅₀ values represented as means±SEM are 7.7± 2.5 nM, 12±2 nM, 8.1±1.1 nM, 9.6±2.5 nM, and 4.1±2.4 nM, respectively. Comparison of EC₅₀ values using a Student’s t-test failed to show any significant (p<0.05) differences between dopamine alone or in the presence of any secondary synthon.

Fig. 5. Radioligand binding competition analyses with full-length D₃ receptor-selective antagonists

Radioligand binding competition assays were performed as described in Section 2. Binding was conducted using [³H]methylspiperone plus the indicated concentrations of (A) sulpiride, (B) PG 571, (C) GCC 3-05, (D) PG 648, (E) JJC 4-077, or (F) NGB 2904. Data are mean graphs expressed as percent specific binding and plotted as mean±SEM, n=3–4. K_i values were calculated from observed IC₅₀ values using the Cheng–Prusoff equation and are displayed in Table 1.

Fig. 6. Radioligand binding saturation analyses in the absence or presence of competing ligands

Radioligand binding assays were performed as described in Section 2. (A) Saturation binding isotherm of [³H]methylspiperone in the absence of any compound (control) or in the presence of 2 nM GCC 3-05, 1 μM sulpiride, or 0.5 nM PG571, as indicated. (B) Scatchard transformation of data in A. K_D and B_max values were determined using GraphPad Prizm: control, 0.21 nM, 5,910 fmol/mg; GCC 3-05, 0.91 nM, 6,590 fmol/mg; sulpiride, 1.13 nM, 6,190 fmol/mg, and PG 571, 2.0 nM, 6,580 fmol/mg. The data shown are from a representative experiment of two independent experiments with similar results, each performed in triplicate.

Fig. 7. Curve–shift analyses of antagonist inhibition of dopamine-stimulated β-arrestin translocation to the D₃ receptor

β-arrestin recruitment assays were performed as described in Section 2. Dopamine concentration response curves were generated in the absence or presence of the indicated concentrations of sulpiride (A), GCC 3-05 (B), or PG 571 (C). The experiment shown is representative of three independent experiments each performed in triplicate and is plotted as the mean relative luminescence units (RLU)±SEM. Representative Schild plots (insets) of the data are shown and Hill slopes were determined using GraphPad Prism from each individual experiment (A–C). Slopes did not differ from unity and were 1.04±0.05, 1.12±0.10, and 1.01±0.10 respectively (n=3–5), expressed as mean±SEM.

Fig. 8. SB269,652 competition analysis for [³H]methylspiperone binding to the D₃ receptor

(A) Structure of SB269,652. (B) Radioligand binding competition assays were performed as described in Section 2 using [³H]methylspiperone plus the indicated concentrations of SB269,652. Data are representative of three experiments run independently and are expressed as a percentage of the specific binding and plotted as mean±SEM. The mean K_i value was calculated from observed IC₅₀ values from individual experiments using the Cheng–Prusoff equation and determined to be 1.5±0.3 nM.

Fig. 9. Radioligand binding saturation analyses in the absence or presence of SB269,652

Radioligand binding assays were performed as described in Section 2. Saturation binding of [³H] methylspiperone in the presence of no drug (control) or 50 nM SB269,652, as indicated. Data are presented in Scatchard coordinates. K_D and B_max values are: control, 1.2 nM, 14 pmol/mg; SB269652, 2.2 nM, 14.5 pmol/mg. The experiment shown is representative of three independent experiments each performed in triplicate.

Fig. 10. SB269,652 antagonism of dopamine-stimulated β-arrestin translocation to the D₃ receptor

β-arrestin recruitment assays were performed as described in Section 2. Cells were stimulated with an EC₉₀ concentration of dopamine (2 μM) and the indicated concentrations of test compound. Inhibitory concentration response curves for SB269,652 and sulpiride are shown as indicated. The experiment shown is representative of three independent experiments each performed in triplicate and are plotted as mean relative luminescence units (RLU) ±SEM. IC₅₀ values were determined from non-linear regressions of individual experiments and found to be 41.8 nM±7.0 nM for sulpiride and 92.4±17 nM SB269,652, n=3.

Fig. 11. Curve-shift analysis of SB269,652 inhibition of dopamine-stimulated β-arrestin translocation to the D₃ receptor

β-arrestin recruitment assays were performed as described in Section 2. Dopamine concentration response curves were generated in the absence or presence of the indicated concentrations of sulpiride (A), or SB269,652 (B). The experiment shown is representative of three independent experiments each performed in triplicate and are plotted as mean relative luminescence units (RLU)±SEM. Representative Schild plots of the data are shown (insets), and Hill slopes were determined using GraphPad Prism from each individual experiment. The Hill slope was found to be 1.04±0.05 for sulpiride and found to be curvilinear for SB269,652.

Fig. 12

Effect of an SB269,652-related secondary synthon on D₃ receptor radioligand binding and dopamine-stimulated β-arrestin translocation. (A) Structure of CS 01-12. (B) Radioligand binding competition assays were performed as described in Section 2. Competition binding was conducted using the indicated concentrations of CS 01-12. Data are representative of three experiments run independently and are expressed as a percentage of the specific binding. (C) β-arrestin recruitment assays were performed as described in Section 2 Cells were stimulated with the indicated concentrations of dopamine either alone, or in the presence of 50 μM CS 01-12, as indicated. Data represent mean±SEM values from three independent experiments run in triplicate and plotted as average relative luminescence units (RLU). EC₅₀ values were determined from non-linear regressions of individual experiments and found to be 12.1±2.3 nM for dopamine alone, and 12.9±3 nM for dopamine +CS 01-12. Comparison of EC₅₀ values using the Student’s t-test failed to show a significant (p<0.05) difference between dopamine alone or in the presence of CS 01-12.