Tamoxifen Directly Interacts with the Dopamine Transporter.

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 µM) and amphetamine-stimulated efflux (59% reduction at 10 µM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.

Nonclassical pharmacology of the dopamine transporter: atypical inhibitors, allosteric modulators, and partial substrates.

The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic, and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. Because both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands possess an addictive liability equivalent to that of cocaine. However, certain recently developed ligands, such as atypical benztropine-like DAT inhibitors with reduced or even a complete lack of cocaine-like rewarding effects, suggest that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classic preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein that can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychological effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H.

In Petunia × hybrida cv 'Mitchell Diploid' (MD), floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is controlled spatially, developmentally, and daily at molecular, metabolic, and biochemical levels. Multiple genes have been shown to encode proteins that either directly catalyse a biochemical reaction yielding FVBP compounds or are involved in metabolite flux prior to the formation of FVBP compounds. It was hypothesized that multiple transcription factors are involved in the precise regulation of all necessary genes, resulting in the specific volatile signature of MD flowers. After acquiring all available petunia transcript sequences with homology to Arabidopsis thaliana R2R3-MYB transcription factors, PhMYB4 (named for its close identity to AtMYB4) was identified, cloned, and characterized. PhMYB4 transcripts accumulate to relatively high levels in floral tissues at anthesis and throughout open flower stages, which coincides with the spatial and developmental distribution of FVBP production and emission. Upon RNAi suppression of PhMYB4 (ir-PhMYB4) both petunia cinnamate-4-hydroxylase (PhC4H1 and PhC4H2) gene transcript levels were significantly increased. In addition, ir-PhMYB4 plants emit higher levels of FVBP compounds derived from p-coumaric acid (isoeugenol and eugenol) compared with MD. Together, these results indicate that PhMYB4 functions in the repression of C4H transcription, indirectly controlling the balance of FVBP production in petunia floral tissue (i.e. fine-tunes).

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

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

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.

Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter.

BACKGROUND: Treatment of stimulant-use disorders remains a formidable challenge, and the dopamine transporter (DAT) remains a potential target for antagonist or agonist-like substitution therapies. METHODS: This review focuses on DAT ligands, such as benztropine, GBR 12909, modafinil, and DAT substrates derived from phenethylamine or cathinone that have atypical DAT-inhibitor effects, either in vitro or in vivo. The compounds are described from a molecular mechanistic, behavioral, and medicinal-chemical perspective. RESULTS: Possible mechanisms for atypicality at the molecular level can be deduced from the conformational cycle for substrate translocation. For each conformation, a crystal structure of a bacterial homolog is available, with a possible role of cholesterol, which is also present in the crystal of Drosophila DAT. Although there is a direct relationship between behavioral potencies of most DAT inhibitors and their DAT affinities, a number of compounds bind to the DAT and inhibit dopamine uptake but do not share cocaine-like effects. Such atypical behavior, depending on the compound, may be related to slow DAT association, combined sigma-receptor actions, or bias for cytosol-facing DAT. Some structures are sterically small enough to serve as DAT substrates but large enough to also inhibit transport. Such compounds may display partial DA releasing effects, and may be combined with release or uptake inhibition at other monoamine transporters. CONCLUSIONS: Mechanisms of atypical DAT inhibitors may serve as targets for the development of treatments for stimulant abuse. These mechanisms are novel and their further exploration may produce compounds with unique therapeutic potential as treatments for stimulant abuse.

The atypical stimulant and nootropic modafinil interacts with the dopamine transporter in a different manner than classical cocaine-like inhibitors.

Modafinil is a mild psychostimulant with pro-cognitive and antidepressant effects. Unlike many conventional stimulants, modafinil has little appreciable potential for abuse, making it a promising therapeutic agent for cocaine addiction. The chief molecular target of modafinil is the dopamine transporter (DAT); however, the mechanistic details underlying modafinil's unique effects remain unknown. Recent studies suggest that the conformational effects of a given DAT ligand influence the magnitude of the ligand's reinforcing properties. For example, the atypical DAT inhibitors benztropine and GBR12909 do not share cocaine's notorious addictive liability, despite having greater binding affinity. Here, we show that the binding mechanism of modafinil is different than cocaine and similar to other atypical inhibitors. We previously established two mutations (W84L and D313N) that increase the likelihood that the DAT will adopt an outward-facing conformational state--these mutations increase the affinity of cocaine-like inhibitors considerably, but have little or opposite effect on atypical inhibitor binding. Thus, a compound's WT/mutant affinity ratio can indicate whether the compound preferentially interacts with a more outward- or inward-facing conformational state. Modafinil displayed affinity ratios similar to those of benztropine, GBR12909 and bupropion (which lack cocaine-like effects in humans), but far different than those of cocaine, ß-CFT or methylphenidate. Whereas treatment with zinc (known to stabilize an outward-facing transporter state) increased the affinity of cocaine and methylphenidate two-fold, it had little or no effect on the binding of modafinil, benztropine, bupropion or GBR12909. Additionally, computational modeling of inhibitor binding indicated that while ß-CFT and methylphenidate stabilize an "open-to-out" conformation, binding of either modafinil or bupropion gives rise to a more closed conformation. Our findings highlight a mechanistic difference between modafinil and cocaine-like stimulants and further demonstrate that the conformational effects of a given DAT inhibitor influence its phenomenological effects.

Regulation of the dopamine transporter: aspects relevant to psychostimulant drugs of abuse.

Dopaminergic signaling in the brain is primarily modulated by dopamine transporters (DATs), which actively translocate extraneuronal dopamine back into dopaminergic neurons. Transporter proteins are highly dynamic, continuously trafficking between plasmalemmal and endosomal membranes. Changes in DAT membrane trafficking kinetics can rapidly regulate dopaminergic tone by altering the number of transporters present at the cell surface. Various psychostimulant DAT ligands-acting either as amphetamine-like substrates or cocaine-like nontranslocated inhibitors-affect transporter trafficking, triggering rapid insertion or removal of plasmalemmal DATs. In this review, we focus on the effects of psychostimulants of addiction (particularly D-methamphetamine and cocaine) on DAT regulation and membrane trafficking, with an emphasis on how these drugs may influence intracellular signaling cascades and transporter-associated scaffolding proteins to affect DAT regulation. In addition, we consider involvement of presynaptic receptors for dopamine and other ligands in DAT regulation. Finally, we discuss possible implications of transporter regulation to the putative toxicity of several substituted amphetamine derivatives commonly used as recreational drugs, as well as to the design of therapeutics for cocaine addiction.