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.
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