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. 2021 Nov 11;12:6505. doi: 10.1038/s41467-021-26757-z

Fig. 2. Development and nanoscale pharmacological characterization of fluo-cariprazine.

Fig. 2

a The docking pose of the fluorescently tagged cariprazine (Fluo-CAR, yellow) in a D3R (gray) homology model (for details, see Supplementary Information). The hydrogen bonds between the key protein residues (Glu2.65, Asp3.32 in red) and the pharmacoprobe are represented as yellow dashed lines. b, c Functional effects of Fluo-CAR on D3R in BRET-based Gi1-activation assay. b Fluo-CAR antagonized the effect of the D3R selective agonist PD128907. Cells were incubated with Fluo-CAR or vehicle for 5 min before PD128907 application. Fluo-CAR pretreatment increased the EC50 value of PD128907 from 1.047 nM to 256.1 nM (n = 3). c Fluo-CAR alone acted as a weak partial agonist. Sigmoidal concentration-response curve was fitted (EC50 = 44.1 nm). BRET ratios are expressed as the percentage of the signal of 1 µM PD128907 (n = 3). d Representative confocal images of HA-D3R-expressing HEK 293 cells treated with Fluo-CAR (100 nM) following HA immunostaining. Fluo-CAR selectively binds those cells that express HA-D3R. e, f Competitive ligand-binding experiment assessed by quantitative dual direct STORM imaging. HA-D3R-expressing cells were incubated with either vehicle (Veh) or 10 µM SB277011-A (SB), a selective D3R antagonist. The ratio of PharmacoSTORM and ImmunoSTORM localization points (LPs) was normalized to vehicle pretreatment. SB277011-A pretreatment markedly reduced the Fluo-CAR signal (n = 4, two-tailed Mann–Whitney U test, P = 0.0286). g Saturation binding assay of Fluo-CAR treatment measured within the plasma membrane. Relative (rel.) receptor occupancy (the ratio of PharmacoSTORM and ImmunoSTORM LPs) was expressed as the percentage of the signal of 1 µM Fluo-CAR, n values are (from left to the right) 4; 4; 3; 4; 3; 3; 3; 7. One-site sigmoidal binding curve was fitted (half-maximal occupancy was at 123 nM). h, i Nanoscale visualization of Fluo-CAR binding sites in the sub-nanomolar concentration range. The representative image is shown in (h). White arrows point to sparsely bound individual Fluo-CAR molecules. i Higher magnification of the lower concentration range from (g) is shown (two-tailed Mann–Whitney U test, P = 0.0294). Data are presented as mean ± SEM, n values indicate biologically independent experiments in all panels.