2,3-Disubstituted quinuclidines as a novel class of dopamine transporter inhibitors

There is considerable interest in developing dopamine transporter (DAT) inhibitors as potential therapies for the treatment of cocaine abuse. We report herein our pharmacophore-based discovery and molecular modeling-assisted rational design of 2,3-disubstituted quinuclidines as potent DAT inhibitors with a novel chemical scaffold. Through 3-D-database pharmacophore searching, compound 12 was identified as a very weak DAT inhibitor with K(i) values of 7.3 and 8.9 microM in [3H]mazindol binding and in inhibition of dopamine reuptake, respectively. Molecular modeling-assisted rational design and chemical modifications led to identification of potent analogues (-)-29 and 34 with K(i) values of 14 and 32 nM for both compounds in binding affinity and inhibition of dopamine reuptake, respectively. Behavioral pharmacological evaluations in rodents showed that 34 has a profile very different from cocaine. While 34 is substantially more potent than cocaine as a DAT inhibitor, it is approximately four times less potent than cocaine in mimicking the discriminative stimulus properties of cocaine in rat. On the other hand, 34 (3-30 mg/kg) lacks either the locomotor stimulant or stereotypic properties of cocaine in mice. Importantly, 34 blocks locomotor stimulant activity induced by 20 mg/kg cocaine in mice, with an estimated ED(50) of 19 mg/kg. Taken together, our data suggest that 34 represents a class of potent DAT inhibitors with a novel chemical scaffold and a behavioral pharmacological profile different from that of cocaine in rodents. Thus, 34 may serve as a novel lead compound in the ultimate development of therapeutic entities for cocaine abuse and/or addiction.     

Molecular modeling, structure--activity relationships and functional antagonism studies of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketones as a novel class of dopamine transporter inhibitors.

We previously disclosed the discovery of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3) as a novel class of dopamine transporter (DAT) inhibitors and showed that (+/-)-3 has a significant functional antagonism against cocaine in vitro. Our previous preliminary structure-activity relationship study led to identification of a more potent DAT inhibitor [(+/-)-4] but this compound failed to show any significant functional antagonism. To search for more potent analogues than 3 but still displaying significant functional antagonism, further SARs, molecular modeling studies and in vitro pharmacological evaluation of this novel class of DAT inhibitors were performed. Sixteen new analogues were synthesized in racemic form and evaluated as DAT inhibitors. It was found that seven new analogues are reasonably potent DAT inhibitors with K(i) values of 0.041--0.30 and 0.052--0.16 microM in [(3)H]mazindol binding and inhibition of DA reuptake. Chiral isomers of several potent DAT inhibitors were obtained through chiral HPLC separation and evaluated as inhibitors at all the three monoamine transporter sites. In general, the (-)-isomer is more active than the (+)-isomer in inhibition of DA reuptake and all the (-)-isomers are selective inhibitors at the DAT site. Evaluation of cocaine's effect on dopamine uptake in the presence and absence of (+)-3 and (-)-3 showed that (-)-3 is responsible for the functional antagonism obtained with the original lead (+/-)-3. Out of the new compounds synthesized, analogue (+/-)-20, which is 8- and 3-fold more potent than (+/-)-3 in binding and inhibition of DA reuptake, appeared to have improved functional antagonism as compared to (+/-)-3.     

Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters

Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.     

Synthesis and biological activity of 2-carbomethoxy-3-catechol-8-azabicyclo[3.2.1]octanes

Cocaine inhibits the dopamine transporter and the consequent elevation of dopamine is thought to contribute to the addictive properties of cocaine. Tropane analogues of cocaine, targeted to the dopamine transporter (DAT), are a significant focus of drug design for cocaine addiction medications. Herein, we report the function of the ortho hydroxy substituents in dopamine with respect to the azabicyclo[3.2.1]octane skeleton. The introduction of the o-dihydroxyl functionality led to reduced binding potency at monoamine transporters, rather than enhanced interaction with the DAT. It is therefore likely that the binding site for these compounds on the DAT is not the same as that for dopamine. Notwithstanding the moderate potency of the free catechols (>100 nM), 7 manifested stimulant activity with a duration of effect that exceeded 4 h in a rat locomotor activity assay. Compound 10, a diacetoxy prodrug for 7, substituted fully for cocaine in a rat drug-discrimination paradigm and is now undergoing further investigation as a potential medication for cocaine abuse.     

Discovery of substituted 3,4-diphenyl-thiazoles as a novel class of monoamine transporter inhibitors through 3-D pharmacophore search using a new pharmacophore model derived from mazindol

Substituted 3,4-diphenyl-1,3-thiazols were identified as a class of novel and potent monoamine transporter inhibitors through a 3-D pharmacophore search using a new pharmacophore model derived from mazindol. The most potent compound (13) has K(i) values of 24 and 23 nM in binding to dopamine transporter and inhibition of dopamine reuptake, respectively.     

2,3-disubstituted 6-azabicyclo[3.2.1]octanes as novel dopamine transporter inhibitors

A series of cis and trans 3beta-aryl-2-carbomethoxy-6-azabicyclo[3.2.1]octanes, with different substitution at the para-position of the aryl group, were synthesized and examined for reuptake inhibition at the dopamine transporter (DAT). The potency for inhibition of DA reuptake was compared with that of cocaine to determine the significance of the replacement of the 8-azabicyclo[3.2.1]octane (tropane nucleus), displayed in cocaine, for the 6-azabicyclo[3.2.1]octane (normorphan framework). This bicyclic core structure constitutes a novel chemical scaffold in DAT inhibitor design, which may provide new insights into the 3D structure of the DAT and its interaction with cocaine and DA. Among these compounds, the trans-amine series 8 were the most potent ligands at the DAT. In particular, the normorphan analogue 8c (bearing a p-chloro substituent at the beta-aryl group, IC(50)=452 nM) displayed a potency that is in the same range as cocaine (IC(50)=459 nM) itself.     

Synthesis and monoamine transporter binding properties of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes

A series of 2beta-[3'-(substituted benzyl)isoxazol-5-yl]- and 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2beta-[3'-(substituted benzyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (3a-h) showed high binding affinities for the dopamine transporter (DAT). The IC(50) values ranged from 5.9 to 22nM. On the other hand, the 2beta-[3'-methyl-4'-(substituted phenyl)isoxazol-5-yl]-3beta-(substituted phenyl)tropanes (4a-h), with IC(50) values ranging from 65 to 173nM, were approximately 3- to 25-fold less potent than the corresponding 2beta-[3'-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3Beta-(4-Methylphenyl)-2beta-[3'-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC(50) of 5.9nM at the DAT and K(i)s of 454 and 113nM at the NET and 5-HTT, respectively, was the most potent and DAT-selective analog. Molecular modeling studies suggested that the rigid conformation of the isoxazole side chain in 4a-h might play an important role on their low DAT binding affinities.     

Synthesis and receptor binding properties of 2beta-alkynyl and 2beta-(1,2,3-triazol)substituted 3beta-(substituted phenyl)tropane derivatives

A series of 2beta-alkynyl and 2beta-(1,2,3-triazol)substituted 3beta-(substituted phenyl)tropanes were synthesized and evaluated for affinities at dopamine, serotonin, and norepinephrine membrane transporters using competitive radioligand binding assays. All tested compounds were found to exhibit nanomolar or subnanomolar affinity for the dopamine transporter (DAT). One of the most potent and selective compounds in the series was 3beta-(4-chlorophenyl)-2beta-(4-nitrophenylethynyl)tropane (10c) that possessed an IC(50) value of 0.9nM at the DAT and K(i) values of 230nM and 620nM at the norepinephrine transporter (NET) and serotonin transporter (5-HTT), respectively.     

Synthesis of 8-thiabicyclo[3.2.1]oct-2-enes and their binding affinity for the dopamine and serotonin transporters

The reinforcing and stimulant properties of cocaine have been primarily associated with its propensity to bind to monoamine transport systems, in particular the dopamine transporter. Inhibition of the dopamine transporter then leads to an increase of synaptic dopamine with substantial pharmacological consequences. The search for medications for cocaine abuse has had a particular focus on tropane analogs of cocaine, and the interchange of nitrogen for oxygen in this class has led to potent and selective inhibitors of monoamine transport. Herein we report that 8-thiatrop-2-enes are highly potent and quite selective inhibitors of the dopamine transporter. The 3,4-dichlorophenyl-8-thiabicyclo[3.2.1]oct-2-ene (4f) is particularly potent (IC₅₀ = 4.5 nM) and selective (800-fold) with respect to inhibition of the serotonin transporter.     

Design and synthesis of an irreversible dopamine-sparing cocaine antagonist

Cocaine is a powerful reinforcer and stimulant that binds to specific recognition sites associated with monoamine transporters in the mammalian brain. The search for a functional antagonist to the addictive properties of cocaine has focused on the discovery of a molecule that can inhibit cocaine binding to the dopamine transporter (DAT) but continue to allow dopamine transport by the DAT. No such dopamine-sparing cocaine antagonist has been reported and it is becoming evident that dopamine-sparing antagonism of the pharmacological effects of cocaine by a classical antagonist may not be possible. Herein we present a new concept for the design of dopamine-sparing cocaine antagonists. A unique approach is utilized to deliver an inhibitor that binds irreversibly to the DAT, then cleaves and leaves behind a small fragment attached to the DAT that blocks access by cocaine but permits dopamine transport. The design of these compounds takes advantage of a cysteinyl sulfhydryl group in the DAT. This group is hypothesized to attack the incoming inhibitor and lead to selective inhibition of the cocaine binding site while sparing dopamine transport. This concept of a mechanism based irreversible dopamine-sparing cocaine antagonist has now been demonstrated to be viable and, as example, the unsaturated 6 showed inhibition of cocaine (63%) at the DAT after 24h incubation, while at that point considerably less inhibition of dopamine is manifested (23%). In contrast, the epoxide 7 showed a greater inhibition of dopamine reuptake than cocaine binding at 24h (68% versus 18%).     

The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter

The binding affinity of the cocaine analog [(3)H]2 beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT.     

Syntheses of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in vivo pharmacological evaluation: identification of inhibitors of the human dopamine transporter

A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human norepinephrine transporter. The binding potency and reuptake inhibition efficacy was compared with that of (-)-cocaine to determine the significance of removing the two-carbon bridge of the cocaine nucleus on the inhibition of transporter binding and reuptake. Of the transporters examined, the substituted piperidines were relatively selective for the human dopamine transporter. In all cases examined, the cis-diastereomer of the 3-carbomethoxy-4-(aryl-substituted)piperidine was observed to be a more potent inhibitor of the human dopamine transporter than the trans diastereomer. Based on the K(i) (binding) and IC(50) (reuptake inhibition) values obtained, the most potent inhibitor of the series was cis-3-carbomethoxy-4-(4'-chlorophenyl)piperidine, and this compound suppressed spontaneous- and cocaine-induced stimulation in non-habituated male Swiss-Webster mice. The conclusion is that substantial portions of the cocaine structure can be dissected away to provide compounds with significant binding and reuptake inhibition of the human dopamine transporter.     

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.     

A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate

Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high-affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69-fold less sensitive to cocaine inhibition when compared with the wild-type mouse DAT. The triple mutation also resulted in a 47-fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine-insensitive DAT mutants can be used to generate a knock-in mouse line to study the role of DAT in cocaine addiction.     

Pharmacophore-based discovery of 3,4-disubstituted pyrrolidines as a novel class of monoamine transporter inhibitors

3,4-Disubstituted pyrrolidines were discovered as a novel class of monoamine transporter inhibitors through 3-D database pharmacophore searching using a new pharmacophore model. The most potent analogue 12 has Ki values of 0.084 microM in [3H]mazindol binding, 0.20, 0.23, and 0.031 microM in inhibition of dopamine (DA), serotonin (SER), and norepinephrine (NE) reuptake, respectively. Functional antagonism testing in vitro showed that 11 and 12 are weak cocaine antagonists.     

Novel diphenylalkyl piperazine derivatives with high affinities for the dopamine transporter

The novel diphenyl piperazine derivatives containing the phenyl substituted aminopropanol moiety, including 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1, which were modified at the connective between the diphenyl and piperazine moieties, have been found to be potent dopamine uptake inhibitors. To study the further structure-activity relationship (SAR) of these compounds, a new series was synthesized, with modifications at the 2-hydroxy-3-phenylaminopropyl moiety of 1. The series was evaluated for dopamine transporter (DAT) binding affinity with [3H]GBR12935 in rat striatal membranes. Most of the compounds showed moderate to high DAT binding affinities and some were approximately equivalent in activity to compound 1 or GBR12909 as a dopamine uptake inhibitor, with IC(50) values of nanomolar range. The SAR suggested that on exhibiting a potent interaction with the DAT, there is probably a steric limitation around the benzene ring of the phenylamino moiety of 1, allowing only small-sized substituents with the exception of basic moieties at the 4-position. In addition, the SAR at the 3-amino-2-propanol moiety of 1 suggested that either the nitrogen atom with an electron donating substituent or the unsubstituted nitrogen atom and also the hydroxy group are desirable for elicitation of a potent DAT binding affinity.     

Further structure-activity relationship studies on 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives at monoamine transporters

The synthesis and structure–activity relationships of 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives were investigated at the dopamine transporter (DAT), the serotonin transporter (SERT) and norepinephrine transporter (NET). The rigid ethylidenyl-8-azabicyclic[3.2.1]octane skeleton imparted modestly stereoselective binding and uptake inhibition at the DAT. Additional structure–activity studies provided a transporter affinity profile that was reminiscent of the structure–activity of GBR 12909. From these studies, the 8-cyclopropylmethyl group has been identified as a unique moiety that imparts high SERT/DAT selectivity. In this study the 8-cyclopropylmethyl derivative 22e (DAT K_i of 4.0 nM) was among the most potent compounds of the series at the DAT and was the most DAT selective ligand of the series (SERT/DAT: 1060). Similarly, the 8-chlorobenzyl derivative 22g (DAT K_i of 3.9 nM) was found to be highly selective for the DAT over the NET (NET/DAT: 1358).     

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuT(Aa) leucine transporter as a template

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.