PMC

DAT diagram and ligands. (A) Two-dimensional schematic representation of the human dopamine transporter (hDAT). Residues relevant to the present study are shown as enlarged dark circles with single letter code in white. (B) Structure of dopamine, cocaine, the cocaine analogue CFT ((−)-2β -carbomethoxy-3β -(4-fluorophenyl)tropane), BZT, AHN 1-055 and JHW007.

Evidence for involvement of Asn157^3.51 in binding of BZT and JHW007. (A, B, C) Inhibition of [³H]dopamine uptake by CFT (A), BZT (B) or JHW007 (C). The uptake experiments were performed using indicated concentrations of inhibitor on COS7 cells transiently expressing wild type (WT) DAT (black circles) or N157A (blue squares). Data are means ± S.E. of 3 to 17 experiments performed in triplicate.

Evidence for involvement of Ala479^10.51 and Ala480^10.52 in binding of BZT and JHW007. (A, B, C) Inhibition of [³H]dopamine uptake by CFT (A), BZT (B) or JHW007 (C). The uptake experiments were performed using indicated concentrations of inhibitor on COS7 cells transiently expressing wild type (WT) DAT (blue circles) or A479V-A480V (red squares). Data are means ± S.E. of 3 to 17 experiments performed in triplicate.

Evidence for involvement of Val152^3.46 in binding of BZT and JHW007. (A) [³H]CFT/CFT competition binding on wild type (WT) DAT (black circles) or V152I (blue square). (B, C) Inhibition of [³H]CFT binding by BZT (B) or JHW007 (C) to wild type DAT (black circles) or I152I (blue square). The binding experiments were performed using indicated concentrations of non-labeled inhibitor on intact COS7 cells transiently expressing WT DAT or V152I. Data are means ± S.E. of 3 to 17 experiments performed in triplicate.

Evidence for involvement of Asn157^3.51, Ala479^10.51 and Ala480^10.52 in binding of AHN 1-055. (A) Molecular model of DAT/AHN1-055 complex. The filtered binding pose of AHN1-055 is similar to that of JHW007 (see ). (B) Inhibition of [³H]dopamine uptake by AHN 1-055 at WT DAT (black circles), N157C (blue triangles) and A479V-A480V (red squares). The uptake experiments were performed using indicated concentrations of inhibitor on COS7 cells transiently expressing indicated constructs. Data are means ± S.E. of 3 experiments performed in triplicate.

Molecular models of DAT/ligand complexes. The aligned structures of JHW007 and BZT in A illustrate their large common moiety. The docking poses of CFT (green), BZT (red) and JHW007 (blue) in DAT are superimposed in B based on the S1 binding site residues. Sodium and chloride ions around the S1 site and residues Asn157^3.51, Ala479^10.51 and Ala480^10.52 are indicated. The representative poses for JHW007 and BZT are depicted individually in C and D. In JHW007 (C) docking model, the polar interaction between Asn157^3.51 and the fluoride substituent is indicated by a dotted line. The absence of this interaction in the BZT docking model (D) allows the ligand to get closer to Ala479^10.51. Panel E shows the side chain interaction fingerprints () of the filtered docking pose clusters of CFT, BZT, JHW007, and AHN1-055. The interactions between the fluoride substituent and Asn157^3.51 are highlighted in orange.