Interaction of cocaine-, benztropine-, and GBR12909-like compounds with wild-type and mutant human dopamine transporters: molecular features that differentially determine antagonist-binding properties

Abstract

The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine's behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [(3)H]CFT binding to wild-type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine) exhibited a cocaine-like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3alpha stereochemistry tended to exhibit benztropine-like binding, whereas those with 3beta stereochemistry were more cocaine-like. Our results point to the importance of specific molecular features--most notably the presence of a diphenylmethoxy moiety--in determining a compound's binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor-transporter interaction could lead to the development of DAT inhibitors with differential clinical utility.

Fig. 1

Diagram of the membrane topology of the human dopamine transporter based upon the bacterial transporter LeuT_Aa crystal structure. The DAT is a member of the neurotransmitter:sodium symporter (NSS) superfamily (also referred to as SLC6) of 12-transmembrane-domain proteins. Full transmembrane helices are numbered 1-12 and smaller non-membrane intra- and extracellular loops are numbered 1-5 prefixed with either ‘i’ or ‘e,’ respectively. TMs 1 and 6 are thought to be critical for both substrate and cocaine binding. The DAT mutants employed in this study (residues highlighted in red) result in an increase in affinity for cocaine and certain other DAT inhibitors.

Fig. 2

Molecular structures of experimental compounds used in this study, as well as other putative dopamine uptake inhibitors: (a) cocaine and related phenyltropane derivatives; (b) benztropine, GBR12909 and other related compounds containing a diphenylmethoxy moiety, as well as bupropion, a compound that exhibits benztropine-like binding properties despite its distinct structure; (c) compounds containing a benzhydryl group, as well as the close analogue methylphenidate. These different families of compounds may interact with different sites on the transporter protein, triggering different downstream and behavioral effects.