The binding sites for benztropines and dopamine in the dopamine transporter overlap

Abstract

Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational modeling together with site-directed mutagenesis to characterize the binding site for BZTs in DAT. Docking into molecular models based on the structure of the bacterial homolog LeuT supported a BZT binding site that overlaps with the substrate-binding pocket. In agreement, mutations of residues within the pocket, including(2) Val152(3.46) to Ala or Ile, Ser422(8.60) to Ala and Asn157(3.51) to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [(3)H]dopamine uptake inhibition assays and/or [(3)H]CFT competition binding assay. A putative polar interaction of one of the phenyl ring fluorine substituents in JHW007 with Asn157(3.51) was used as a criterion for determining likely binding poses and establish a structural context for the mutagenesis findings. The analysis positioned the other fluorine-substituted phenyl ring of JHW007 in close proximity to Ala479(10.51)/Ala480(10.52) in transmembrane segment (TM) 10. The lack of such an interaction for BZT led to a more tilted orientation, as compared to JHW007, bringing one of the phenyl rings even closer to Ala479(10.51)/Ala480(10.52). Mutation of Ala479(10.51) and Ala480(10.52) to valines supported these predictions with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine.

Fig. 1

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.

Fig. 2

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.

Fig. 3

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.

Fig. 4

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 (Deng et al., 2004) 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.

Fig. 5

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.

Fig. 6

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 Fig. 4). (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.