Pharmacological targeting of G protein-coupled receptor heteromers

Abstract

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D₁ and D₃ receptors (D₁R and D₃R) influences the pharmacological properties of three structurally similar selective dopamine D₃R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D₁R-D₃R heteromer-disrupting peptides, it could be demonstrated that the three D₃R ligands display different D₁R-D₃R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D₁R-mediated signaling in the D₁R-D₃R heteromer; PG01037, acting as a D₃R antagonist cross-antagonized D₁R-mediated signaling in the D₁R-D₃R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D₁R-D₃R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D₃R ligands that are dependent on D₁R-D₃R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D₃R ligands in vivo. The results supported the involvement of D₁R-D₃R heteromers in the locomotor activation by D₁R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D₁R-D₃R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Keywords: Dopamine D(1) receptor; Dopamine D(3) receptor; G protein-coupled receptor (GPCR) heteromers; L-DOPA-induced dyskinesia; Locomotor activation; Mouse; PG01037 (PubChem CID: 11477180); PG01042 (PubChem CID: 11443078); Pramipexole (PubChem CID: 119570); Rat; SKF81297 (PubChem CID: 1218); VK4–116 (PubChem CID: 130431318).

Figure 1.. Differential effects of PG01042, PG01037, and VK4-116 on G protein-dependent signaling in HEK-293T cells transfected with D₃R and D₁R.

a-e. Results from cAMP formation experiments in HEK-293T cells transfected with D₁R-Rluc cDNA (1 μg) with or without D₃R-YFP cDNA (1.5 μg) (D₁R-D₃R cells and D₁R cells, respectively). In a, cells are treated with the D₂-like receptor agonist pramipexole (D3Rago; 30 nM for 10 min) or the D₃R ligands PG01042, PG01037 and VK4-116 (all at 10 or 100 nM for 15 min) before forskolin (Fk, 0.5 μM). In b-e, cells are pre-treated or not with D₁R TM6 or TM7 peptides (4 μM for 4 h) and treated with PG01042 (10 nM), PG01037 (10 nM) and VK4-116 (100 nM) for 15 min before the D₁R agonist SKF81297 (30 nM; D1Rago). Values of cAMP formation are shown as mean ± S.D. (n = 6) and expressed as percentage of Fk-treated or D1Rago-treated cells in each condition (100% represents 80–100 pmols cAMP/10⁶ cells). ****: p < 0.0001 versus FK; ### and ####: p < 0.001 and p < 0.0001 versus D1Rago, respectively (one-way ANOVA followed by Tukey’s post hoc comparisons). f. Results from BiFC experiments in HEK-293T cells co-transfected with D₁R-nYFP and D₃R-cYFP in the absence (−) or the presence of the indicated TM peptides (at 4 μM) from D₁R (gray symbols and plots) or D₃R (blue symbols and plots). Fluorescence values (in means ± S.D.) are expressed as the percentage of the fluorescence in the absence (−) of the indicated TM peptides (n = 6, with triplicates); ***: p < 0.001 versus control values (one-way ANOVA followed by Dunnett’s post hoc comparisons). The schemes illustrate extracellular and parallel to the membrane views of the computational model of the D₁R-D₃R heteromer built using the TM 5/6 interface (see text). g-i. Representative structures (solid sticks) and evolution (lines) of PG01042 (g, in green), PG01037 (h, in orange) and VK4-116 (i, in purple) in complex with D₃R (white cylinders, only the initial structure is shown) as devised from three replicas of unbiased 1μs MD simulations. The second pharmacophore unit of PG01042 and VK4-116 remained stable at the ECD near TM 2, whereas this part of PG01037 favors its interaction with TM 6 (see Suppl. Fig. 2).

Figure 2.. Differential effects of PG01042, PG01037, and VK4-116 on β-arrestin recruitment in HEK-293T cells transfected with D₃R and D₁R.

a-c. Results from β-arrestin recruitment experiments in HEK-293T cells transfected with β-arrestin-1-Rluc cDNA (0.5 μg), D₃R-YFP cDNA (1 μg cDNA) with or without D₁R cDNA (1.5 μg cDNA) (D₁R-D₃R cells and D₃R cells, respectively). In a-b, cells are treated for 10 min with the D₂-like receptor agonist pramipexole (D3Rago; 30 nM) or the D₃R ligands PG01042, PG01037 and VK4-116 (10 or 100 nM). In c, cells are pre-treated or not with D₁R TM6 or TM7 peptides (4 μM for 4 h) and with VK4-116 (10 or100 nM) for 15 min before the D₁R agonist SKF81297 (30 nM; D1Rago). Coelenterazine H (5 μM) was added before pramipexole or the selective D₃R ligands for 7 minutes and β-arrestin-1 recruitment was measured by BRET (see Material and Methods). Values are mean ± S.D. (n = 8). *, *** and ****: p < 0.05, p < 0.001 and p < 0.0001 versus basal, respectively (one-way ANOVA followed by Dunnett’s post hoc comparisons). d. Evolution of the C_α atoms (spheres) of Y32^1.35 in TM 1, L89^2.64 in TM 2, I101^3.23 in TM 3, F170^4.62 in TM 4, F188^5.38 in TM 5, H354^6.60 in TM 6, and P362^7.32 in TM 7 during three replicas of unbiased 1 μs MD simulations of the D₃R monomer (gray) and the D₁R-D₃R heteromer (blue) with no ligand bound. Contour plots of the distances between Y32^1.35 in TM 1 and F188^5.38 in TM 5 (distance TM1-TM5) and between L89^2.64 in TM 2 and H354^6.60 in TM 6 (distance TM2-TM6) during the MD simulations. Distributions of these distances are shown in the axes. e. Evolution of I183^ECL2 (spheres), and contour plots and distributions of X,Z coordinates corresponding to the C_b atom of I183^ECL2 during MD simulations. Black arrows represent the movements of D₃R in the D₁R-D₃R heteromer (blue) relative to the D₃R protomer (gray). f. Detailed view of I183^ECL2 of D₃R in the D₁R-D₃R heteromer during MD simulations with no ligand bound (blue) and PG01042 (green), PG01037 (orange) and VK4-116 (red) bound to D₃R. Distributions of the Z coordinate corresponding to the C_α atom of I183^ECL2 during MD simulations. The ethyl group of VK4-116 that triggers the upward movement of I183^ECL2 is highlighted. g. Detailed views of I183^ECL2, F188^5.38, S192^5.42, and T115^3.37 of D₃R in the D₁R-D₃R heteromer during MD simulations with no ligand bound (blue) and PG01042 (green), PG01037 (orange) and VK4-116 (red) bound to D₃R. Frequency contacts (%) between side-chain residues, color-coded according to the ligand bound to D₃R, as calculated with the GetContacts software. Black arrows represent the movements of these side chains of D₃R relative to the unliganded D₃R (blue). The xy plane is as defined by the Orientations of Proteins in Membranes (OPM) [73].

Figure 3.. Differential effects of PG01042, PG01037, and VK4-116 on the locomotor activation induced by D₁R and D₂-like receptor agonists in reserpinized mice.

Mice were administered reserpine (5 mg/kg, s.c.) 20 h before administration of the D₁R agonist SKF 81297 (SKF; 5 mg/kg, i.p.), the D₂-like receptor agonist pramipexole (PMX; 1, 3 or 10 mg/kg, i.p.), or both, without (a) or with (b) previous administration (15 min before) of the D₃R ligands PG01042 (10 mg/kg, i.p.), PG01037 (30 mg/kg, i.p.) or VK4-116 (10 mg/kg, i.p.). Values are mean ± S.E.M. (n = 5–18) and are expressed as the average of the transformed counts (squared root) obtained from the 10 min-periods recorded for 1 h. In a, **, *** and ****: p < 0.01, p < 0.001 and p < 0.0001 versus vehicle-treated animals (represented by the dotted line), respectively; ###: p < 0.001 versus SKF (one-way ANOVA followed by Tukey’s post hoc comparisons) (one-way ANOVA followed by Tukey’s post hoc comparisons). In b, *, **, *** and ****: p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 versus respective saline-treated animals, respectively.

Figure 4.. D₁R, D₂R, and D₃R gene (DRD1, DRD2 and DRD3) expression in the mouse striatum.

a. RNAscope ISH results in the ventral striatum (framed area in the coronal section) showing strong expression of DRD1, DRD2 and DRD3 mRNA with a strong co-localization of DRD3 and DRD1 and limited expression of DRD3 in DRD2-expressing neurons. b. RNAscope ISH results in the dorsal striatum (framed area in the coronal section) showing a strong expression of DRD1 and DRD2 mRNA and limited expression of DRD3 mRNA, which predominates in DRD1-expressing neurons. Images were taken under 60X magnification. DRD1 in red, DRD2 in blue, DRD3 in green.

Figure 5.. Differential effects of PG01042, PG01037, and VK4-116 on L-DOPA-induced dyskinesia.

In a within-subjects, counterbalanced design, hemiparkinsonian rats previously rendered dyskinetic were treated with the D₃R ligands (a) PG01042 (0, 5, 10 mg/kg; i.p.), (b) PG01037 (0, 10, 30 mg/kg; i.p.), or (c) VK4-116 (VK; 0, 10, 30 mg/kg; i.p.) prior to administration of L-DOPA (6 mg/kg; s.c.). Rats were rated for Axial, Limb and Orolingual (ALO) abnormal involuntary movements (AIMs) every 10 min for 180 min post-injection. AIMs time course and ALO sums are expressed as median + median absolute deviation (M.A.D.). Data were analyzed by non-parametric Friedman ANOVAs with Wilcoxon Match Pairs post-hoc tests. + p < 0.05 for Vehicle versus low dose, # p < 0.05 for vehicle versus high dose, $ p < 0.05 for low dose versus high dose.