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2-Substituted 3?-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

ABSTRACT: Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3?-aryltropanes with 2?-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2?-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2?-Ph2COCH2-3?-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2? compounds increased locomotion, only the 2?-(4-ClPh)PhCOCH2-3?-4-Cl-Ph analog had cocaine-like efficacy. None of the 2?-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2?- and 2?-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

SUBMITTER: Hong WC

PROVIDER: S-EPMC4767397 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Project description:The dopamine transporter (DAT), a member of the neurotransmitter:Na(+) symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end.Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates.Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

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2-Substituted 3?-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

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