Design of a True Bivalent Ligand with Picomolar Binding Affinity for a G Protein-Coupled Receptor Homodimer

Abstract

Bivalent ligands have emerged as chemical tools to study G protein-coupled receptor dimers. Using a combination of computational, chemical, and biochemical tools, here we describe the design of bivalent ligand 13 with high affinity ( K_DB1 = 21 pM) for the dopamine D₂ receptor (D₂R) homodimer. Bivalent ligand 13 enhances the binding affinity relative to monovalent compound 15 by 37-fold, indicating simultaneous binding at both protomers. Using synthetic peptides with amino acid sequences of transmembrane (TM) domains of D₂R, we provide evidence that TM6 forms the interface of the homodimer. Notably, the disturber peptide TAT-TM6 decreased the binding of bivalent ligand 13 by 52-fold and had no effect on monovalent compound 15, confirming the D₂R homodimer through TM6 ex vivo. In conclusion, by using a versatile multivalent chemical platform, we have developed a precise strategy to generate a true bivalent ligand that simultaneously targets both orthosteric sites of the D₂R homodimer.

Figure 1.

A) Components for the design of bivalent ligands. B) Bivalent ligands (12–13) and their corresponding monovalent counterparts (14–15).

Scheme 1.

^a Synthesis of the bivalent ligands 12 and 13 and the monovalent ligands 14 and 15. ^a Reagents and conditions: (a) 10, EDC·HCl, HOBt·H₂O, DIEA, DMF, rt, 16 h (49%); (b) 11, EDC·HCl, HOBt·H₂O, DIEA, DMF, rt, 16 h (64%); (c) EDC·HCl, dry DMF, rt, 2 h, then 8, DIEA, dry DMF, rt, 90 min (79%); (d) 10, EDC·HCl, HOBt·H₂O, DIEA, DMF, rt, 16 h (25%); (e) EDC·HCl, dry DMF, rt, 2 h, then 9, DIEA, dry DMF, rt, 90 min (80%); (f) 11, EDC·HCl, HOBt·H₂O, DIEA, DMF, rt, 16 h (24%).

Figure 2.

Antagonistic effect of the studied compounds on global cellular response induced by sumanirole. Dynamic mass redistribution (DMR) assays were performed in CHO cells stably expressing D₂R and A_2AR. A) Quantification of the antagonist effect of all D₂R ligands on DMR. Values are mean±SEM from 3 determinations carried out in triplicates. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s post hoc test. **p<0.01 compared to sumanirole alone. B) Representative DMR curves from one of these experiments in which cells were treated with medium (control) (black), with 1μM of spiperone (grey), with 1μM of monovalent compounds 14 (light blue) or 15 (dark blue) or with 500 nM of bivalent compounds 12 (orange) or 13 (red) for 30 minutes. After that, cells were treated with 100 nM of sumanirole. Each curve is the mean of a representative optical trace experiment carried out in triplicates. The resulting shifts of reflected light wavelength (pm) were monitored over time. C) Dose-response of the antagonistic effect of bivalent compound 13 (black) (IC₅₀=15±3 nM) and monovalent 15 (red) (IC₅₀=280±70 nM) on the DMR induced by 100 nM sumanirole. Data are mean±SEM from 3–8 experiments and are presented as percentage of the maximal effect of sumanirole.

Figure 3.

Effect of TAT-TM peptides on disturbance of the D₂R homodimer, determined by BiFC experiments in HEK-293T cells transfected with D₂R-nYFP and D₂R-cYFP cDNA. Values are mean±SEM from 6–9 determinations. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s post hoc test. ***p<0.001 compared to non TAT-TM treated complementation.

Figure 4.

Effect of TAT-TM6 peptides of D₂R and A_2AR on competition experiments of [³H]YM-09151–2 vs. D₂R ligands. Competition curves with increasing concentrations of monovalent 15 (in A) or bivalent 13 (in B) D₂R ligands in the absence (black) or in the presence of TAT-TM6 of A_2AR (red) or TAT-TM6 of D₂R (blue), using membranes from sheep brain striatum. Data are mean±SEM from 3 experiments performed in triplicate.

Figure 5.

Evolution of bivalent ligand 13 in the D₂R homodimer (TM1 and TM4, ECL1 and part of ECL2 are omitted for clarity), constructed via the TM6 interface, as devised from MD simulations. The structures of 13 (the color code of the atoms is as in Figure 1) are extracted from the simulations (20 structures collected every 50 ns), whereas the structure of the D₂R homodimer corresponds to the initial model. A detailed analysis of the simulation (Figure S3) confirms that the designed bivalent ligand 13 remains stable at the orthosteric binding cavities through the unbiased 1 μs MD simulation.