Figure2_final_R1_v2.pdf

Cocaine and αPVP enantiomers binding mode in DAT. (A) Mechanism of action of competitive and non-competitive (allosteric vs. atypical) inhibition. (B) Dissociation of [3H]WIN35428 in DAT membranes. Data are mean ± SEM n ≥ 3 independent experiments conducted in duplicates. (C) Single-point uptake inhibition of 0.2 µM [3H]DA conducted in HEK293cells stably expressing DAT. Compounds are pre-incubated at their IC50 alone or in combinations (Ø =control, untreated; C = cocaine 100 nM; S = S-αPVP 10 nM; S/C = cocaine 100 nM + S-αPVP 10 nM; R = R-αPVP 300 nM; R/C = R-αPVP 300 nM + cocaine 100 nM). One-way ANOVA followed by Dunnett´s multiple comparison test vs. cocaine *=P < 0.05, **=P < 0.01, ***=P < 0.001. (C–F) DAT-WT and DAT-F76Y uptake inhibition profiles for cocaine, S-αPVP and R-αPVP. (G) Site-directed mutagenesis of DAT orthosteric binding site. DAT residues are mutated in the corresponding residue in SERT. Data are shown as Ki fold change compared to WT DAT (mean ± SD). Every symbol represents an individual experiment conducted in triplicate. (H) Membrane and front view of the S1 site of the human DAT showing the best docking pose of S-αPVP (blue) and the binding pose of cocaine elucidated by X-Ray in dDAT (PDB: 4XP4) (orange). TM10 and TM11 are not shown for clarity, while residues F76, S149, V152, and S429 are highlighted as sticks and colored by atom type. The two sodium ions are represented as dark blue spheres. (I) 2D interaction map of S-αPVP with the human DAT according to the binding mode shown in panel H.