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ABSTRACT: This report describes development of a series of novel bivalent molecules with a pharmacophore derived from the D2/D3 agonist 5-OH-DPAT. Spacer length in the bivalent compounds had a pronounced influence on affinity for D2 receptors. A 23-fold increase of D2 affinity was observed at a spacer length of 9 or 10 (compounds 11d and 14b) compared to monovalent 5-OH-DPAT (Ki; 2.5 and 2.0 vs. 59 nM for 11d and 14b vs. 5-OH-DPAT, respectively). Functional potency of 11d and 14b indicated a 24- and 94-fold increase in potency at the D2 receptor compared to 5-OH-DPAT (EC50; 1.7 and 0.44 vs. 41 nM for 11d and 14b vs. 5-OH-DPAT, respectively). These are the most potent bivalent agonists for D2 receptor known to date. This synergism is consonant with cooperative interaction at the two orthosteric binding sites in the homodimeric receptor.
ACS Medicinal Chemistry Letters 10/2012; 3(12):991-996. · 3.36 Impact Factor
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ABSTRACT: To further explore the basic structural motifs (3S,6S)-6-benzhydryl-N-benzyltetrahydro-2H-pyran-3-amine and (2S,4R,5R)-2-benzhydryl-5-(benzylamino)tetrahydro-2H-pyran-4-ol, developed by our research group, for monoamine transport inhibition, we designed and synthesized various structurally altered analogues. The new compounds were tested for their affinities for the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET) in rat brain by measuring their capacity to inhibit the uptake of [(3) H]DA, [(3) H]5-HT, and [(3) H]NE, respectively. Our results point to novel compounds with a TUI, DNRI, SNRI, or SSRI profile. Among the TUIs, compound 2 g exhibited a balanced potency for all three monoamine transporters (K(i) : 60, 79, and 70.3 nM for DAT, SERT, and NET, respectively). In the rat forced swim test, compound 2 g produced a significant decrease in immobility in drug-treated rats relative to vehicle, indicating a potential antidepressant property.
ChemMedChem 10/2012; · 3.15 Impact Factor
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ABSTRACT: The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared with non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl-β-cyclodextrin (mβCD), which effectively depletes total membrane cholesterol--uniformly affecting cholesterol-DAT interactions in both raft and non-raft membrane domains--reduced both DA uptake and efflux rate. In contrast, disruption of raft-localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft-localized DAT in substrate uptake or efflux. Supranormal repletion of cholesterol-depleted cells with the analog desmosterol, a non-raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn(2+) and the conformationally biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward-facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol-DAT interactions in regulating DAT function.
Journal of Neurochemistry 09/2012; · 4.06 Impact Factor
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ABSTRACT: In our effort to develop multifunctional drugs against Parkinson's disease, a structure-activity-relationship study was carried out based on our hybrid molecular template targeting D2/D3 receptors. Competitive binding with [(3)H]spiroperidol was used to evaluate affinity (K(i)) of test compounds. Functional activity of selected compounds in stimulating [(35)S]GTPγS binding was assessed in CHO cells expressing either human D2 or D3 receptors. Our results demonstrated development of highly selective compounds for D3 receptor (for (-)-40K(i), D3 = 1.84 nM, D2/D3 = 583.2; for (-)-45K(i), D3 = 1.09 nM, D2/D3 = 827.5). Functional data identified (-)-40 (EC(50), D2 = 114 nM, D3 = 0.26 nM, D2/D3 = 438) as one of the highest D3 selective agonists known to date. In addition, high affinity, nonselective D3 agonist (-)-19 (EC(50), D2 = 2.96 nM and D3 = 1.26 nM) was also developed. Lead compounds with antioxidant activity were evaluated using an in vivo PD animal model.
Journal of Medicinal Chemistry 05/2012; 55(12):5826-40. · 4.80 Impact Factor
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ABSTRACT: The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25°C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4°C for 3h, [Na(+)] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.
Journal of neuroscience methods 09/2011; 203(1):19-27. · 2.30 Impact Factor
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ABSTRACT: Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 μM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 μM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 μM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 μM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.
Journal of Neurochemistry 06/2011; 118(5):714-20. · 4.06 Impact Factor
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ABSTRACT: The dopamine D3 receptor has been implicated as a potential target for drug development in various complex psychiatric disorders including psychosis, drug dependence, and Parkinson's disease. In our overall goal to develop molecules with preferential affinity at D3 receptors, we undertook a hybrid drug development approach by combining a known dopamine agonist moiety with a substituted piperazine fragment. In the present study, three compounds produced this way with preferential D3 agonist activity, were tested at D3 receptors with mutations in the agonist binding pocket of three residues known to be important for agonist binding activity. At S192A and T369V, the hybrid agonist compounds produced an interaction profile in [(3)H]spiperone binding assays similar to that of the parent 5-OH-DPAT and 7-OH-DPAT molecules. The loss of affinity at the S192A mutant was most prominent for 5-OH-DPAT and its corresponding hybrid compound D237. D110N did not show any radioligand binding. Homology modeling indicated that 7-OH-DPAT-derived D315 uniquely shares H-bonding with Tyr365 which produced favorable interaction and no loss of H-bonding in the S192A mutant, suggesting that agonist activity may not be solely controlled by residues in the binding pocket.
Biochemical pharmacology 01/2011; 81(1):157-63. · 4.25 Impact Factor
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ABSTRACT: 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, β-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 β-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.
PLoS ONE 01/2011; 6(10):e25790. · 4.09 Impact Factor
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Manju A Kurian,
Yan Li,
Juan Zhen,
Esther Meyer,
Nebula Hai,
Hans-Jürgen Christen,
Georg F Hoffmann,
Philip Jardine,
Arpad von Moers,
Santosh R Mordekar, [......],
Elizabeth Wraige,
Christa Dietrich,
Timothy Lewis,
Keith Hyland,
Simon Heales,
Terence Sanger,
Paul Gissen,
Birgit E Assmann, Maarten E A Reith,
Eamonn R Maher
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ABSTRACT: dopamine transporter deficiency syndrome is the first identified parkinsonian disorder caused by genetic alterations of the dopamine transporter. We describe a cohort of children with mutations in the gene encoding the dopamine transporter (SLC6A3) with the aim to improve clinical and molecular characterisation, reduce diagnostic delay and misdiagnosis, and provide insights into the pathophysiological mechanisms.
11 children with a biochemical profile suggestive of dopamine transporter deficiency syndrome were enrolled from seven paediatric neurology centres in the UK, Germany, and the USA from February, 2009, and studied until June, 2010. The syndrome was characterised by detailed clinical phenotyping, biochemical and neuroradiological studies, and SLC6A3 mutation analysis. Mutant constructs of human dopamine transporter were used for in-vitro functional analysis of dopamine uptake and cocaine-analogue binding.
children presented in infancy (median age 2·5 months, range 0·5-7) with either hyperkinesia (n=5), parkinsonism (n=4), or a mixed hyperkinetic and hypokinetic movement disorder (n=2). Seven children had been initially misdiagnosed with cerebral palsy. During childhood, patients developed severe parkinsonism-dystonia associated with an eye movement disorder and pyramidal tract features. All children had raised ratios of homovanillic acid to 5-hydroxyindoleacetic acid in cerebrospinal fluid, of range 5·0-13·2 (normal range 1·3-4·0). Homozygous or compound heterozygous SLC6A3 mutations were detected in all cases. Loss of function in all missense variants was recorded from in-vitro functional studies, and was supported by the findings of single photon emission CT DaTSCAN imaging in one patient, which showed complete loss of dopamine transporter activity in the basal nuclei.
dopamine transporter deficiency syndrome is a newly recognised, autosomal recessive disorder related to impaired dopamine transporter function. Careful characterisation of patients with this disorder should provide novel insights into the complex role of dopamine homoeostasis in human disease, and understanding of the pathophysiology could help to drive drug development.
The Lancet Neurology 01/2011; 10(1):54-62. · 23.46 Impact Factor
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ABSTRACT: A successful replacement pharmacotherapy for treating cocaine dependency would likely reduce cocaine's abuse, support a low abuse liability, overlap cocaine's subjective effects, and have a long duration of action. Inhibitors with varying selectivity at the dopamine transporter (DAT) have approximated these properties. The objective of the present study was to characterize the behavioural effects of an extremely selective DAT inhibitor, (+) trans-4-(2-Benzhydryloxyethyl)-1-(4-fluorobenzyl) piperadin-3-ol (D-84), a 3-hydroxy substituted piperidine derivative of GBR-12935, for its cocaine-like discriminative stimulus effects, its effects on cocaine self-administration, and for its own self-administration. During cocaine discrimination tests, cocaine occasioned the 10 mg/kg cocaine training stimulus with an ED(50) value of 3.13 (1.54-6.34) mg/kg, and reduced response rates with an ED(50) value of 20.39 (7.24-57.44) mg/kg. D-84 incompletely generalized to the cocaine stimulus occasioning a maximal 76% cocaine-lever responding, while reducing response rates with lower potency than cocaine (ED(50)=30.94 (12.34-77.60) mg/kg). Pretreatment with D-84 (9.6-30.4 mg/kg) significantly (P<0.05) reduced cocaine intake at 17.1 mg/kg D-84 when cocaine was self-administered at 0.5 mg/kg/infusion, and at 30.4 mg/kg D-84 when cocaine was self-administered at 0.1, 0.5 .and 1.0 mg/kg/infusion. During self-administration tests with D-84 (0.1-1 mg/kg/infusion), numbers of infusions significantly exceeded vehicle levels at 0.3 mg/kg/infusion. These results show that D-84 pretreatment can decrease cocaine intake especially when high doses of cocaine are being self-administered. This observation, combined with its incomplete generalization to the cocaine discriminative stimulus and its reported long duration of action, provides a profile consistent with a potential replacement therapy for treating cocaine-abusing patients.
European journal of pharmacology 12/2010; 648(1-3):127-32. · 2.59 Impact Factor
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ABSTRACT: Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) DAT with W84L mutant, effects of d-amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d-amphetamine to reduce surface Myc upon co-expressing Flag-WT with Myc-W84L but not Myc-WT with Flag-W84L, and from the sluggish Myc-W84L endocytosis rate (both with or without d-amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which D-amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher-order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co-expression and antibody reversal in western blotting. Upon co-transfecting Myc- and Flag-tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species.
Journal of Neurochemistry 08/2010; 114(3):873-85. · 4.06 Impact Factor
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ABSTRACT: In receptor binding assays with ultra-high-affinity radioligands, it is difficult, in practice, to adhere the golden rule that the receptor concentration in the assay should be substantially (at least 10-fold) lower than the dissociation constant (K(d)) of the radioligand and inhibition constant (K(i)) of compound. Especially for low specific activity radioligands (usually tritiated ligands of a couple of TBq/mmol), routinely applied in concentrations at around or below the K(d), the use of extremely small amounts of receptor protein per assay will result in low levels of bound radioactivity; the alternative use of larger assay volumes will make it difficult to apply 96-well filtration devices. For assessing the inhibition constant (K(i)) of competitive inhibitors under conditions violating the above golden rule, equations are available incorporating both [receptor] and [ligand] versus K(d); however, their application requires precise knowledge of [receptor] or initial bound/free [radioligand] ratio. In this study, we present the theoretical basis for determining the K(i) for a competitive inhibitor in a new protocol at high [protein] and high [radioligand] with the simple Cheng-Prusoff correction without the need to correct for [receptor] or initial bound/free [radioligand] ratio. In addition, we present results on the binding of the ultra-high-affinity ligand [(3)H]spiperone to dopamine D(2) and D(3) receptors validating the K(i) values calculated with the new protocol for competitive inhibitors as compared with those calculated with the most comprehensive equation available to date, that of Munson and Rodbard (1988). Binding was measured at varying [radioligand] and [receptor], test compounds (including (-)5-OH-DPAT, (+/-)7-OH-DPAT, and ropinirole) were run with varying [receptor], and simulations were done at vastly varying [radioligand] for inhibitors with vastly different K(i)s. The modified high [radioligand] protocol presented here removes a major hindrance in the proper execution of binding assays with ultra-high-affinity tritiated ligands with K(d) values in the sub-nanomolar range, allowing the use of 96-well plates with small volumes of 100-200 microl per binding assay.
Journal of neuroscience methods 04/2010; 188(1):32-8. · 2.30 Impact Factor
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ABSTRACT: 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.
Journal of Neurochemistry 03/2010; 112(6):1605-18. · 4.06 Impact Factor
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ABSTRACT: Here we report structure-activity relationship study of a novel hybrid series of compounds where structural alteration of aromatic hydrophobic moieties connected to the piperazine ring and bioisosteric replacement of the aromatic tetralin moieties were carried out. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. SAR results identified compound (-)-24c (D-301) as one of the lead molecules with preferential agonist activity for D3 receptor (EC(50) (GTP gamma S); D3 = 0.52 nM; D2/D3 (EC(50)): 223). Compounds (-)-24b and (-)-24c exhibited potent radical scavenging activity. The two lead compounds, (-)-24b and (-)-24c, exhibited high in vivo activity in two Parkinson's disease (PD) animal models, reserpinized rat model and 6-OHDA induced unilaterally lesioned rat model. Future studies will explore potential use of these compounds in the neuroprotective therapy for PD.
Journal of Medicinal Chemistry 02/2010; 53(3):1023-37. · 4.80 Impact Factor
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ABSTRACT: The role of iron in the pathogenesis of Parkinson's disease (PD) has been implicated strongly because of generation of oxidative stress leading to dopamine cell death. In our overall goal to develop bifunctional/multifunctional drugs, we designed dopamine D2/D3 agonist molecules with a capacity to bind to iron. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptor with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds was carried out with GTPgammaS binding assay. SAR results identified compounds (+)-19a and (-)-19b as two potent agonists for both D2 and D3 receptors (EC(50) (GTPgammaS); D2 = 4.51 and 1.69 nM and D3 = 1.58 and 0.74 nM for (-)-19b and (+)-19a, respectively). In vitro complexation studies with 19b demonstrated efficient chelation with iron. Furthermore, the deoxyribose assay with 19b demonstrated potent antioxidant activity. In PD animal model study, (-)-19b exhibited potent in vivo activity in reversing locomotor activity in reserpinized rats and also in producing potent rotational activity in 6-OHDA lesioned rats. This reports initial development of unique lead molecules that might find potential use in symptomatic and neuroprotective treatment of PD.
Journal of Medicinal Chemistry 02/2010; 53(5):2114-25. · 4.80 Impact Factor
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ABSTRACT: 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.
Annals of the New York Academy of Sciences 02/2010; 1187:316-40. · 3.15 Impact Factor
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ABSTRACT: 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.
Annals of the New York Academy of Sciences 01/2010; 1187(1):316 - 340. · 3.15 Impact Factor
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ABSTRACT: Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [(3)H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K(i)) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (-)-10e (K(i); D2=47.5 nM, D3=0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K(i); D2=113 nM, D3=3.73 nM). Additionally, compound (-)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPgammaS binding study, one of the lead molecules, (-)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3.
Bioorganic & medicinal chemistry 07/2009; 17(11):3923-33. · 2.82 Impact Factor
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Manju A Kurian,
Juan Zhen,
Shu-Yuan Cheng,
Yan Li,
Santosh R Mordekar,
Philip Jardine,
Neil V Morgan,
Esther Meyer,
Louise Tee,
Shanaz Pasha,
Evangeline Wassmer,
Simon J R Heales,
Paul Gissen, Maarten E A Reith,
Eamonn R Maher
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ABSTRACT: Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.
The Journal of clinical investigation 07/2009; 119(6):1595-603. · 15.39 Impact Factor
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ABSTRACT: A novel series of optically active molecules based on a 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.Herein we describe the synthesis and biological evaluation of a series of asymmetric 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol-based dihydroxy compounds in which the hydroxy groups are located on both the piperidine ring and the N-phenylethyl side chain. In vitro uptake inhibition data of these molecules indicate high affinity for the dopamine transporter (DAT) in addition to moderate to high affinity for the norepinephrine transporter (NET). Interestingly, compounds 9 b and 9 d exhibit affinities for all three monoamine transporters, with highest potency at DAT and NET, and moderate potency at the serotonin transporter (SERT) (K(i): 2.29, 78.4, and 155 nM for 9 b and 1.55, 14.1, and 259 nM for 9 d, respectively). Selected compounds 9 a, 9 d, and 9 d' were tested for their locomotor activity effects in mice and for their ability to occasion the cocaine-discriminative stimulus in rats. These test compounds generally exhibit a much longer duration of action than cocaine for elevating locomotor activity, and completely generalize the cocaine-discriminative stimulus in a dose-dependent manner.
ChemMedChem 06/2009; 4(7):1075-85. · 3.15 Impact Factor
