Dissociation of high-affinity cocaine analog binding and dopamine uptake inhibition at the dopamine transporter

Cocaine initiates its euphoric effects by binding to the dopamine transporter (DAT), blocking uptake of synaptic dopamine. It has been hypothesized that the DAT transmembrane aspartic acid residue D79 forms an ionic interaction with charged nitrogen atoms in both dopamine and cocaine. We examined the consequences of novel and previously studied mutations of the D79 residue on DAT uptake of [3H]dopamine, DAT binding of the cocaine analog [3H]WIN 35,428, and drug inhibition of each process, all under identical conditions. The rat D79E DAT mutation decreased dopamine uptake Vmax by 7-fold and decreased dopamine turnover by 4-fold. Wild-type DAT displayed near-perfect agreement in the uptake and binding inhibition potencies for substrates, but cocaine and other nonsubstrate inhibitor drugs were approximately 3-fold less potent in uptake than in binding assays. Apparent affinities for substrates were unaffected by the D79E mutation unless the catechol moiety was modified. Strikingly, potencies for nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT, because of a 3-fold lowering of binding affinities relative to WT DAT. The present findings reveal a complex role for D79 in determining substrate specificity and high-affinity binding of DAT inhibitors. We propose that at least two discrete inhibitor-binding DAT conformations or populations exist and that the DAT conformation/population responsible for inhibitor high-affinity binding is less responsible for dopamine uptake. The findings may be extensible to other psychostimulants and antidepressants that display discrepancies between binding affinity and monoamine uptake inhibition potency and may be relevant to development of a long-sought "cocaine antagonist".     

Dual incorporation of photoaffinity ligands on dopamine transporters implicates proximity of labeled domains

We have recently developed novel high-affinity blockers for the dopamine transporter (DAT) by carrying out structure-activity studies of GBR 12909 molecule piperidine analogs. To investigate the molecular basis of binding of these compounds in comparison to known sites of action of GBR 12909, cocaine, and benztropine analogs, we developed a piperidine-based photoaffinity label [(125)I]4-[2-(diphenylmethoxy)ethyl]-1-[(4-azido- 3-iodophenyl)methyl]-piperidine [(125)I]AD-96-129), and used proteolysis and epitope-specific immunoprecipitation to identify the protein domains that interact with the ligand. [(125)I]AD-96-129 became incorporated into two different regions of the DAT primary sequence, an N-terminal site containing transmembrane domains (TMs) 1 to 2, and a second site containing TMs 4 to 6. Both of these regions have been identified previously as sites involved in the binding of other DAT photoaffinity labels. However, in contrast to the previously characterized ligands that showed nearly complete specificity in their binding site incorporation, [(125)I]AD-96-129 became incorporated into both sites at comparable levels. These results suggest that the two domains may be in close three-dimensional proximity and contribute to binding of multiple uptake blockers. We also found that DATs labeled with [(125)I]AD-96-129 or other photoaffinity labels displayed distinctive sensitivities to proteolysis of a site in the second extracellular loop, with protease resistance related to the extent of ligand incorporation in the TM4 to 6 region. These differences in protease sensitivity may indicate the relative proximity of the ligands to the protease site or reflect antagonist-induced conformational changes in the loop related to transport inhibition.     

Structure-activity relationship comparison of (S)-2beta-substituted 3alpha-(bis[4-fluorophenyl]methoxy)tropanes and (R)-2beta-substituted 3beta-(3,4-dichlorophenyl)tropanes at the dopamine transporter

Extensive structure-activity relationships at the dopamine transporter (DAT) have been developed around two classes of tropane-based ligands. Opposing stereoselectivity and divergent structural requirements for optimal DAT binding suggest that these tropane-based DAT inhibitors may not access identical binding domains. To further investigate this hypothesis, a series of (S)-2beta-carboalkoxy-3alpha-(bis[4-fluorophenyl]methoxy)tropanes (11a-f, 13-16) and their identically (R)-2beta-substituted 3beta-(3,4-dichlorophenyl)tropanes (3, 5a-d) were prepared and evaluated for binding at the DAT and for inhibition of [(3)H]dopamine uptake in rat brain. These studies showed that most of the identically 2-carboalkoxy-substituted analogues, within the two classes of compounds, bind with high affinity to DAT (K(i) = 5.5-100 nM), albeit with opposite stereochemistry. However, the larger azido- (15) and isothiocyanato- (16) (S)-2beta-carbophenylethoxy-3alpha-(bis[4-fluorophenyl]methoxy)tropanes demonstrated a significant decrease in DAT binding potency (IC(50) = 210 and 537 nM, respectively), suggesting that the DAT does not tolerate 2-position steric bulk in the benztropine class, as it does with the 2-substituted 3-aryltropanes. In addition, binding affinities at the serotonin transporter, norepinephrine transporter, and muscarinic receptors were evaluated and compared for compounds 2, 3, 11a-e, and 13. Together, the binding profiles across these systems demonstrated significant differences between these two classes of tropane-based ligands, which may be exploited toward the discovery of a cocaine-abuse pharmacotherapeutic.     

Synthesis of 3-arylecgonine analogues as inhibitors of cocaine binding and dopamine uptake

3-Arylecgonine analogues were synthesized and characterized by 1H and 13C NMR, IR, and MS. The compounds were synthesized as racemates from cycloheptatriene-7-carboxylic acid or enantiomerically from (-)-cocaine. These analogues were tested for their ability to inhibit [3H]cocaine binding to bovine striatal tissue and to inhibit [3H]dopamine uptake into striatal synaptosomes. Methyl (1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-ca rboxylate was the most potent analogue. IC50 values for inhibition of cocaine binding and dopamine uptake were 20 and 100 nM, respectively. The racemates and the 1R isomers were equally potent inhibitors of binding and uptake. Methyl (1RS-2-endo-3-exo)-3-(2,4-dinitrophenyl)-8-methyl-8-azabicyclo[3.2 .1]octane- 2-carboxylate was the least potent. IC50 for inhibition of both binding and uptake was 40 microM.     

Isothiocyanate derivatives of cocaine: irreversible inhibition of ligand binding at the dopamine transporter

Isothiocyanate derivatives of (-)-cocaine were prepared and tested for inhibitory potency at the cocaine receptor in rat striatal membranes. Coincubation with m-isothiocyanatobenzoylecgonine methyl ester (m-ISOCOC), p-isothiocyanatobenzoylecgonine methyl ester (p-ISOCOC), and 3 beta-(4-isothiocyanatophenyl)tropane-2-carboxylic acid methyl ester (ISOWIN) resulted in inhibition of [3H]WIN 35,428 binding, but the compounds were about 10-fold weaker than (-)-cocaine. However, p-ISOCOC was approximately 3-fold more potent than metaphit, an isothiocyanate derivative of phencyclidine. p-ISOCOC was equipotent at the serotonin transporter but was much less potent at the norepinephrine transporter and was inactive at the D2 dopamine receptor at 1000 microM concentration. The IC50 value for m-ISOCOC and p-ISOCOC varied with tissue concentration, suggesting irreversible inhibition of binding. Preincubation with m-ISOCOC and p-ISOCOC resulted in inhibition of [3H]WIN 35,428 binding that could not be removed by washing of the membranes; in contrast, preincubation with (-)-cocaine caused inhibition that was readily removed by washing. Preincubation with 1 microM concentrations of p-ISOCOC resulted in a large reduction in Bmax of the high affinity binding site for [3H]WIN 35,428. Preincubation with 100 microM p-ISOCOC eliminated the high affinity site and apparently reduced the affinity at the low affinity site. Coincubation of 10 microM p-ISOCOC with 100 microM cocaine prevented the total loss of [3H]WIN 35,428 binding. The uptake of [3H]dopamine was inhibited by p-ISOCOC with an IC50 comparable to that of cocaine. Additionally, preincubation of rat striatal synaptosomes with 10 microM p-ISOCOC reduced the Vmax of [3H]dopamine uptake after washing. These data suggest that m-ISOCOC and p-ISOCOC are useful irreversible acylators of (-)-cocaine binding sites at the dopamine transporter.     

Labeling of dopamine transporter transmembrane domain 1 with the tropane ligand N-[4-(4-azido-3-[125I]iodophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane implicates proximity of cocaine and substrate active sites

The novel photoaffinity ligand N-[4-(4-azido-3-(125)I-iodophenyl)-butyl]-2-beta-carbomethoxy-3beta-(4-chlorophenyl) tropane ([(125)I]MFZ 2-24) was used to investigate the site for cocaine binding on the dopamine transporter (DAT). [(125)I]MFZ 2-24 irreversibly labeled both rat striatal and expressed human DAT with high affinity and appropriate pharmacological specificity. Tryptic proteolysis of [(125)I]MFZ 2-24 labeled DAT followed by epitope-specific immunoprecipitation demonstrated that the ligand becomes adducted almost exclusively to transmembrane domains (TMs) 1-2. Further localization of [(125)I]MFZ 2-24 incorporation achieved by proteolyzing labeled wild-type and methionine mutant DATs with cyanogen bromide identified the sequence between residues 68 and 80 in TM1 as the ligand adduction site. This is in marked contrast to the previously identified attachment of the photoaffinity label [(125)I]RTI 82 in TM6. Because [(125)I]MFZ 2-24 and [(125)I]RTI 82 possess identical tropane pharmacophores and differ only in the placement of the reactive azido moieties, their distinct incorporation profiles identify the regions of the protein adjacent to different aspects of the cocaine molecule. These findings thus strongly support the direct interaction of cocaine on DAT with TM1 and TM6, both of which have been implicated by mutagenesis and homology to a bacterial leucine transporter as active sites for substrates. These results directly establish the proximity of TMs 1 and 6 in DAT and suggest that the mechanism of transport inhibition by cocaine involves close interactions with multiple regions of the substrate permeation pathway.     

2-Carbomethoxy-3-aryl-8-bicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters

Cocaine is a potent central nervous system stimulant with severe addiction liability. Its reinforcing and stimulant properties derive from inhibition of monoamine transport systems, in particular the dopamine transporter (DAT). This inhibition results in an increase in synaptic dopamine with subsequent stimulation of postsynaptic dopamine receptors. A wide variety of ligands manifest potent inhibition of the DAT, and these ligands include 3-aryltropane as well as 8-oxa-3-aryltropane analogues of cocaine. There has been considerable effort to determine structure-activity relationships of cocaine and congeners, and it is becoming clear that these inhibitors do not all interact with the DAT in the same manner. The functional role of the 8-heteroatom is the focus of this study. We describe the preparation and biology of a series of 2-carbomethoxy-3-arylbicyclo[3.2.1]octane analogues. Results show that methylene substitution of the amine or ether function of the 8-hetero-2-carbomethoxy-3-arylbicyclo[3.2.1]octanes yields potent inhibitors of monoamine transport. Therefore neither nitrogen nor oxygen are prerequisites for binding of tropane-like ligands to monoamine transporters.     

Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo

1. Chronic treatment with low doses of the selective monoamine oxidase (MAO) type B inhibitors selegiline [(-)-deprenyl] and rasagiline, causes elevation in extracellular level of 3,4-dihydroxyphenylethylamine (dopamine) in the rat striatum in vivo (Lamensdorf et al., 1996). The present study was carried out to determine whether this effect of selegiline could be the result of an inhibition of the high-affinity dopamine neuronal transport process. 2. Changes in activity of the dopamine transporter (DAT) in vivo following selegiline treatment were evaluated indirectly by microdialysis technique in the rat, from the change in striatal dopamine extracellular concentration following systemic amphetamine administration (4 mg kg(-1), i.p.). Striatal levels of the DAT molecule were determined by immunoblotting. Uptake of [3H]-dopamine was determined in synaptosomes from selegiline-treated animals. 3. Amphetamine-induced increase in striatal extracellular dopamine level was attenuated by one day and by chronic (21 days) treatment with selegiline (0.25 mg kg(-1), s.c.). 4. Striatal levels of DAT were elevated after 1 and 21 days treatment with selegiline, but were not affected by clorgyline, rasagiline, nomifensine or amphetamine. 5. The increase in DAT expression, and attenuation of amphetamine-induced dopamine release, were not accompanied by a change in [3H]-dopamine uptake in synaptosomes of selegiline-treated animals. 6. The results suggest that a reversible inhibition of dopamine uptake occurs following chronic low dose selegiline treatment in vivo which may be mediated by an increase in endogenous MAO-B substrates such as 2-phenylethylamine, rather than by the inhibitor molecule or its metabolites. Increased DAT expression appears to be a special property of the selegiline molecule, since it occurs after one low dose of selegiline, and is not seen with other inhibitors of MAO-A or MAO-B. The new DAT molecules formed following selegiline treatment appear not to be functionally active.     

Synthesis and ligand binding of cocaine isomers at the cocaine receptor

The cocaine binding site at the dopamine transporter has been found to be stereoselective. Thus, the seven possible stereoisomers of (-)-cocaine have been synthesized and found to inhibit [3H]-2 beta-carbomethoxy-3 beta-(4-fluoro-phenyl)tropane [( 3H]WIN 35,428) with potencies ranging from 1/60 to 1/600 of that of (-)-cocaine. The synthesis and characterization of all new compounds is presented.     

Novel tropane-based irreversible ligands for the dopamine transporter.

3alpha-(diphenylmethoxy)tropane (benztropine) and its analogues are tropane ring-containing dopamine uptake inhibitors that display binding and behavioral profiles that are distinct from cocaine. We previously prepared a benztropine-based photoaffinity label [125I]-(N-[4-(4'-azido-3'-iodophenyl)butyl]-3alpha-[bis(4'-fluorophenyl)methoxy]tropane, [125I]1, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] 2 (RTI 82). To characterize further these different binding domains, photoaffinity ligands that had the 4'-azido-3'-iodophenyl substituent extended from the same position on the tropane ring were desirable. Thus, identification of the optimal alkyl linker between this substituent and the tropane nitrogen in the benztropine series was investigated to ultimately prepare the identical N-substituted analogue of 2. In this pursuit, the N-[4-(4'-azido-3'-iodophenyl)propyl] analogue of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (9a) was synthesized as well as two isothiocyanate analogues that do not require photoactivation (10a,b) for irreversible binding. The synthesis of these target compounds was achieved using a modification of the strategy developed for 1. Evaluation of these compounds for displacing [3H]WIN 35 428 binding at DAT in rat caudate putamen revealed that the 4'-azido-3'-iodophenylbutyl substituent, found in 1, provided optimal binding affinity and was chosen to replace the N-CH3 group on 2. Both the 4'-azido-3'-iodophenyl- and the 4'-isothiocyanatophenylbutyl analogues of 2 (25 and 26, respectively) were synthesized. Both products bound to DAT with comparable potency (IC(50) = 30 nM) to RTI 82 (2). In addition, compound 26 demonstrated wash-resistant displacement of [3H]WIN 35 428 in HEK 293 cells stably transfected with hDAT. These ligands will provide important tools for further characterizing the binding domains for tropane-based dopamine uptake inhibitors at the DAT.     

Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine- and rimcazole-based inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [(3)H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.     

The dopamine transporter: a vigilant border control for psychostimulant action

Neurotransmission within the mesocorticolimbic dopamine system has remained the central focus of investigation into the molecular, cellular and behavioral properties of psychostimulants for nearly three decades. The primary means by which dopamine transmission in the synapse is terminated is via the dopamine transporter (DAT), the presynaptic plasmalemmal protein that is responsible for the reuptake of released dopamine. Numerous abused as well as clinically important drugs have important pharmacological interactions with DAT. In general, these compounds fall into two categories: those that block dopamine transport (e.g., cocaine, methylphenidate) and those that serve as substrates for transport [e.g., dopamine, amphetamine and 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy")]. Recent data from in vitro and in vivo studies have suggested that DAT, like other biogenic amine transporters, share several characteristics with classical ligand-gated ion channels. In addition, substrates for transport promote redistribution of DAT away from the plasma membrane, while transport inhibitors such as cocaine disrupt this process. In addition, presynaptic autoreceptors for dopamine have been implicated in the modulation of DAT surface expression and function. The present chapter summarizes some of the recent discoveries pertaining to the electrogenic properties of DAT and their potential relevance to the effects of amphetamine-like stimulants on DAT function. Although there are a number of intracellular and extracellular modulatory influences on dopamine clearance that may play particular roles in psychostimulant action, we specifically focus on the differential direct modulation of DAT function by transport substrates and inhibitors, and we also discusses the role of presynaptic D2 receptors in transport regulation.     

Structure-activity relationship studies on a novel series of (S)-2beta-substituted 3alpha-[bis(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues for in vivo investigation

In general, 3alpha-(diphenylmethoxy)tropane (benztropine)-based dopamine uptake inhibitors do not demonstrate cocaine-like pharmacological activity in models of psychostimulant abuse and have been proposed as potential medications for the treatment of cocaine addiction. However, several (S)-2-carboalkoxy-substituted-3alpha-[bis(4-fluorophenyl)methoxy]tropane analogues were discovered to stimulate locomotor activity and substitute in subjects trained to discriminate cocaine, suggesting a role of the 2-position substituent in mediating these cocaine-like actions. Herein, we describe the synthesis of a series of novel N- and 2-substituted-3alpha-[bis(4-fluoro- or 4-chlorophenyl)methoxy]tropane analogues. Most of these analogues demonstrated high affinity binding to the dopamine transporter (DAT; K(i) = 1.8-40 nM), and selectivity over the other monoamine transporters and muscarinic M(1) receptors. When the (S)-2-carboalkoxy substituent was replaced with (S)-2-ethenyl, the resulting analogue 11 demonstrated the highest DAT binding affinity in the series (K(i) = 1.81 nM) with DAT selectivity over serotonin transporters (SERT; 989-fold), norepinephrine transporters (NET; 261-fold) and muscarinic receptors (90-fold). When the 4'-F groups of compounds 5 (K(i) = 2.94 nM) and 8 (K(i) = 6.87 nM) were replaced with 4'-Cl in the (S)-2-carboalkoxy series, DAT binding affinities were slightly reduced (K(i) = 12.6 and 14.6 nM for 6 and 7, respectively), yet inhibition of dopamine uptake potency remained comparably high (IC(50) range = 1.5-2.5 nM). Interestingly, the 4'-Cl analogue (+/-)-6 substituted less in rats trained to discriminate cocaine than the 4'-F analogue (+/-)-5. These studies demonstrate that manipulation of the 2-, N-, and 3-position substituents in the 3alpha-(diphenylmethoxy)tropane class of dopamine uptake inhibitors can result in ligands with high affinity and selectivity for the DAT, and distinctive in vivo pharmacological profiles that cannot be predicted by their effects in vitro.     

Synthesis, monoamine transporter binding, properties, and functional monoamine uptake activity of 3beta-[4-methylphenyl and 4-chlorophenyl]-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes

Synthetic methods were developed for the synthesis of the 3beta-(4-substituted phenyl)-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes (4a-s). The compounds were evaluated for their monoamine transporter binding and monoamine uptake inhibition properties using both rat brain tissue and cloned transporter assays. In general, the compounds showed higher dopamine transporter (DAT) affinity relative to the serotonin and norepinephrine transporters (SERT and NET, respectively) and greater [3H]dopamine uptake inhibition potency relative to [3H]serotonin and [3H]norepinephrine uptake inhibition. Several compounds were DAT selective relative to the SERT and NET in the monoamine transporter binding assays. The most potent and selective analog in the functional monoamine uptake inhibition test was 3beta-(4-methylphenyl-2 beta-[5-(3-nitrophenyl)thiazol-2-yl]tropane (4p).     

Interactions of cocaine with primary and secondary recognition sites on muscarinic receptors.

Several lines of evidence have suggested that muscarinic receptors may possess more than one ligand binding site. In this study, the interactions of cocaine with primary and secondary (allosteric) sites on muscarinic receptors in membrane homogenates from post-mortem human brainstem were examined. (-)-Cocaine inhibited the binding of the tritiated muscarinic antagonists N-methylscopolamine (NMS) and pirenzepine to an apparent single class of sites, with Ki values of 200-300 microM. The binding of the muscarinic agonist [3H]oxotremorine-M was inhibited with a similar Ki value (200 microM). (+)-Cocaine, although not the naturally occurring stereoisomer, was 10-20-fold more potent than (-)-cocaine in competing for binding to the primary muscarinic recognition site. The binding of cocaine was unaffected by guanine nucleotides or N-ethylmaleimide, consistent with its purported action as a competitive antagonist. Cocaine was not selective for muscarinic receptor subtypes. Rosenthal analysis of the [3H]NMS saturation binding data in the presence of increasing concentrations of either (-)-cocaine or (+)-cocaine indicated that both isomers produced an apparent competitive-like reduction in the [3H]NMS affinity. Schild regression analysis of the saturation binding data resulted in curvilinear plots suggestive of cooperative or allosteric interactions of (-)-cocaine with the [3H]NMS-labeled receptors. The effects of (-)-cocaine on the kinetics of [3H]NMS binding were consistent with an allosteric interaction with the receptor. Increasing concentrations of cocaine markedly slowed the rate of [3H]NMS dissociation from the primary recognition site. The allosteric modulation of [3H] NMS binding by (-)-cocaine was abolished with increasing ionic strength. Taken together, these data demonstrate that (-)-cocaine interacts with primary and allosteric recognition sites on muscarinic receptors.     

Cocaine displaces [3H]WIN 35,428 binding to dopamine uptake sites in vivo more rapidly than mazindol or GBR 12909

Previous studies have shown that [3H] WIN 35,428 binds preferentially to striatal cocaine receptors at the dopamine transporter after in vivo injection. In vivo binding competition studies were carried out to assess the relative rates of entry and occupancy of cocaine receptors by (-)-cocaine, mazindol and GBR 12909. After i.v. injection, (-)-cocaine occupied receptors relatively more rapidly than GBR 12,909 while mazindol was the slowest.     

Structure-activity relationships at monoamine transporters for a series of N-substituted 3alpha-(bis[4-fluorophenyl]methoxy)tropanes: comparative molecular field analysis, synthesis, and pharmacological evaluation

The development of structure-activity relationships (SAR) with divergent classes of monoamine transporter ligands and comparison of their effects in animal models of cocaine abuse have provided insight into the complex relationship among structure, binding profiles, and behavioral activity. Many 3alpha-(diphenylmethoxy)tropane (benztropine) analogues are potent dopamine uptake inhibitors but exhibit behavioral profiles that differ from those of cocaine and other compounds in this class. One of the most potent and dopamine transporter (DAT) selective N-substituted benztropine analogues (N-(4-phenyl-n-butyl)-3alpha-(bis[4-fluorophenyl]methoxy)tropane, 1c) is devoid of cocaine-like behaviors in rodent models but is also highly lipophilic (cLogD = 5.01), which compromises its water solubility and may adversely affect its pharmacokinetic properties. To further explore the SAR in this series and ultimately to design dopamine uptake inhibitors with favorable lipophilicities for drug development, a comparative molecular field analysis (CoMFA) was performed on a set of benztropine analogues previously synthesized in our laboratory. The CoMFA field analysis on the statistically significant (r2(cv) = 0.632; r2(ncv) = 0.917) models provided valuable insight into the structural features required for optimal binding to the DAT, which was used to design a series of novel benztropine analogues with heteroatom substitutions at the tropane N-8. These compounds were evaluated for binding at DAT, serotonin (SERT) and norepinephrine (NET) transporters, and muscarinic M1 receptors in rat brain. Inhibition of [3H]DA uptake in synaptosomes was also evaluated. Most of the analogues showed high DAT affinity (12-50 nM), selectivity (10- to 120-fold), potent inhibition of dopamine uptake, and lower lipophilicities as predicted by cLogD values.     

Design and synthesis of 2- and 3-substituted-3-phenylpropyl analogs of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine: role of amino, fluoro, hydroxyl, methoxyl, methyl, methylene, and oxo substituents on affinity for the dopamine and serotonin transporters

Novel derivatives of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909, 1) and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935, 2) with various substituents in positions C2 and C3 of the phenylpropyl side chain were synthesized and evaluated for their ability to bind to the dopamine transporter (DAT) and the serotonin transporter (SERT). In the C2 series, the substituent in the S-configuration, with a lone-pair of electrons, significantly enhanced the affinity for DAT, whereas the steric effect of the substituent was detrimental to DAT binding affinity. In the C3 series, neither the lone electron pair nor the steric effect of the substituent seemed to affect DAT binding affinity, while sp (2) hybridized substituents had a detrimental effect on affinity for DAT. In the series, the 2-fluoro-substituted (S)-10 had the highest DAT binding affinity and good DAT selectivity, while the 2-amino-substituted (R)-8 showed essentially the same affinity for DAT and SERT. The oxygenated 16 and 18 possessed the best selectivity for DAT.     

D2 receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP(+)) with live cell imaging techniques to identify the role of two D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP(+) accumulation. D(2S)R activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP(+) accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D(2)R stimulation. These experiments demonstrate that D(2S)R stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.