Fig. 3. Binding of the bitopic FOB02-04A to the D₃R receptor.

A Schematic of dopamine, pramipexole, rotigotine and the bitopic FOB02-04A ligand shown as sticks and colored by component. B Binding of the secondary pharmacophore (SP) (sticks, dark red) to a groove-shaped pocket at the D₃R (yellow, surface representation) formed by ECL1 and TM1. C Two views of a comparison of FOB02-04A (dark red carbon, sticks), pramipexole (green carbon, sticks), and rotigotine (cyan carbon, sticks) binding into the D₃R pocket (yellow, surface representation). Dashed circles indicate OBS, established SBP, and the SBP_2-ECL1-1 site. D Overall binding mode of the bitopic molecule to the D₃R and ordering of TM1 upon bitopic binding. FOB02-04A (dark red, sticks) is displayed on superposed structures of D₃R bound to eticlopride (cartoon, cyan) and FOB02-04A (cartoon, yellow) E Schematic of the FOB02-04A binding into the D₃R ligand binding pocket. F Binding details of FOB02-04A (dark red, sticks) at the D₃R (yellow sticks) with cryo-EM density as gray mesh. G Binding details of FOB02-04A (dark red, sticks) at the D₃R (yellow cartoons) with residues at the ligand binding pocket colored by functional effect when mutated to alanine: decreased efficacy – green carbons, decreased potency – blue carbon and non-detectable binding – red carbon. H pEC₅₀ values for alanine mutation of the residues at the ligand binding site in response to G_OA activation by FOB02-04A using the TRUPATH assay. All data are means ± SEM of four independent experiments (n = 4) performed in technical triplicates except for D110^3.32A, S196^5.46A, Y365^7.35A, T369^7.39A, W342^6.48A and Y373^7.43A for which there was n = 3, WT, V86^2.61A, L89^2.64A, E90^2.65A, ∆G94^ECL1, F346^6.52A for there was n = 5, and L119^3.41W, T115^3.37A for which there was n = 6. *p < 0.05 (one-way ANOVA with Dunnett post hoc analysis) for D₂R (p = 0.0081), V111^3.33A (p = 0.0049), T115^3.37A (p = 0.0074) and I183^45.82A (p = 0.0013) and nd - non-detectable. I Concentration-response curve of D₃R ΔG94^ECL1 upon G_OA activation by quinpirole (light blue, n = 4) and FOB02-04A (deep blue, n = 5) (shown as net BRET). All data are means ± SEM of the specified biological replicates, each performed in technical triplicates. J Emax values for alanine mutation of the residues at the ligand binding site in response to G_OA activation by FOB02-04A using the TRUPATH assay. Emax values have been normalized to D₃R WT. All data are means ± SEM of four independent experiments performed in technical triplicate (n = 4) except for D110^3.32A, S196^5.46A, Y365^7.35A, T369^7.39A, W342^6.48A, Y373^7.43A (n = 3), WT, V86^2.61A, L89^2.64A, E90^2.65A, ∆G94^ECL1, F346^6.52A (n = 5) and L119^3.41W, T115^3.37A (n = 6). *p < 0.05 (Holm-Sidak multiple comparisons tests two-tailed p value) for H29^1.32A (p = 0.016), V86^2.61A (p = 0.026), F106^3.28A (p = 0.003), F346^6.52A (p = 0.019). K Concentration-response curves of D₃R H29^1.32A (orange), H29^1.32F (pink), H29^1.32K (yellow), and H29^1.32R (green) upon G_OA activation by FOB02-04A (shown as net BRET). All data are means ± SEM derived from three independent experiments (n = 3), each performed in technical triplicate except for H29^1.32A (n = 4). All source data within this figure is provided as a Source Data file.