N-Methyl Amine-substituted Fluoxetine Derivatives - Springer Link

Arch Pharm Res Vol 32, No 12, 1663-1671, 2009 DOI 10.1007/s12272-009-2201-2

N-Methyl Amine-substituted Fluoxetine Derivatives: New Dopamine Transporter Inhibitors Young Sil Yoon1,2, Taesup Cho1,3, Sung-Hwa Yoon4, Churl Ki Min1,4, and Changho Lee5 1 Department of Biological Sciences, Ajou University, Suwon 443-749, Korea, 2Department of Biochemistry, SungKyunKwan University, School of Medicine, Suwon 440-746, Korea, 3Brain Research Center, University of British Columbia, Vancouver, BC V6T 2B5, Canada, 4Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea, and 5 Department of Pharmacology and Biomedical Science, College of Medicine, Hanyang University, Seoul 133-791, Korea

(Received January 21, 2008/Revised August 28, 2009/Accepted August 29, 2009)

Transport of dopamine (DA) by the dopamine transporter from the synaptic cleft into the presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. The binding of psychostimulants to their recognition sites on the DA transporter leads to an inhibition of DA transport and a subsequent rising of the dopamine contents in the synaptic cleft is ascribed to a mode of psychostimulation. Discovery of dopamine transporter inhibitors would be useful with regard to substituting for cocaine and minimizing its abuse. Recently, a number of fluoxetine analogues were synthesized, especially focusing on the substitution of N-methyl amine group through modifying the structure of the fluoxetine, N-methyl-3-[p-trifluoromethylphenoxy]-3-phenylpropylamine, widely used as an antidepressant. Among them, the pharmacological properties of FD-2, (R)-N-ethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine and FD-4, N-(R)-3-trifluorophenoxy-3-phenylpropane-imidazole with a higher affinity for the DA transporter were characterized in terms of dopamine transporter inhibition expecting for useful cocaine substitutes. Effects of the compounds on [H3]dopamine uptake, [I125]RTI-55 binding, and DA transporter-associated currents were examined with the ligand binding assays and voltage clamping technique in human embryonic kidney (HEK)-293 cells where the recombinant human DA transporter (hDAT) was stably expressed. Our results showed that (i) fluoxetine was potent in inhibiting both the uptake of [H3]DA (IC50 = 0.21 ± 0.032 mM, n = 3) and the [I125]RTI-55 binding (IC50 = 0.23 ± 0.012 mM, n = 10); (ii) N-methyl amine substituted fluoxetine analogues, FD-2 and FD-4 were equally or more potent than fluoxetine itself in terms of inhibition of [H3]DA uptake (IC50 FD-2: 0.077 ± 0.0032 mM (n = 3); FD-4: 0.26 ± 0.13 mM (n = 3), inhibition of [I125]RTI-55 binding, and reduction in DA transporter-associated currents, suggesting that these analogues could be a new class of dopamine transporter inhibitors. Key words: Dopamine, Dopamine transporter, Fluoxetine analogues, Dopamine transporter inhibitors

INTRODUCTION

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Correspondence to: Changho Lee, Department of Pharmacology, College of Medicine, Hanyang University, Seoul 133-791, Korea Tel: 82-2-2220-0655, Fax: 82-31-2292-6686 E-mail: [email protected] Churl K. Min, Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea Tel: 82-31-219-2621 E-mail: [email protected]

Neurotransmitter is a chemical that is secreted from the presynaptic terminal, diffuses across the synaptic cleft, binds to its receptor on the postsynaptic cell membrane, and evokes a change in postsynaptic membrane potential. The presence of dopamine (DA) transporter in the dopaminergic presynaptic membrane was identified (Kuhar et al., 1973; Iversen, 1975), and subsequent cloning of cDNA and heterologous expression in Xenopus oocyte suggests that DA transporter belongs to biogenic amine transporter

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family that shares common structural and functional features such as 12 transmembrane domains and Na+ and Cl--dependent transport of its substrate (Giros et al., 1991; Kilty et al., 1991; Shimada et al., 1991; Li and Reith, 2000; for review, see Amara and Kuhar, 1993). DA transporter has been known to provide binding sites for many psychostimulants such as cocaine (Reith and Costa, 1992; Earles and Schenk, 1999), amphetamine (Sitte et al., 1998; Eshleman et al., 1999), mazindol, methylphenidate, GBR12909 (Andersen, 1989; Tatsumi et al., 1997; Inazu, 1999), and RTI-55 (Boja et al., 1992). Binding of such psychostimulants to their binding sites on the DA transporter leads to an inhibition of DA transport and thus a subsequent rising in the concentration of DA in the synaptic cleft, which is ascribed to a mode of psychostimulation (Harris and Balessarini, 1973; Heikkila et al., 1975). In particular, the dopamine transporter (DAT) has been suggested to be the critical recognition site for cocaine, thereby mediating its abuse liability (Ritz et al., 1987; Kuhar et al., 1991) Thus, the discovery of dopamine transporter inhibitors would be useful with regard to substituting for cocaine and minimizing its abuse. In fact, the substitution drug therapy has been highly effective for treating nicotine and heroine addictions. Recently, we synthesized a number of fluoxetine analogues, especially focusing on the substitution of N-methyl amine group through modifying the structure of the fluoxetine, N-methyl-3-[p-trifluoromethylphenoxy]-3-phenylpropylamine, a specific inhibitor of the serotonin (5-HT) transporter (Tomas et al., 1987; Fuller et al., 1991), and widely used as an antidepressant (Fig. 1). Among them, the pharmacological properties of FD-2, (R)-N-ethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine and FD-4, N-(R)-3-trifluorophenoxy-3-phenylpropane-imidazole with a higher affinity for the DA transporter were characterized in terms of dopamine transporter inhibition expecting for useful cocaine substitutes. Effects of the compounds on [H3]dopamine uptake, [I125]RTI-55 binding, and DA transporter-associated currents were examined with the ligand binding assays and voltage clamping technique in human embryonic kidney (HEK)-293 cells where the recombinant human DA transporter (hDAT) was stably expressed.

MATERIALS AND METHODS Chemicals The fluoxetine analogues, including FD-2 ((R)-Nethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine)

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Fig. 1. Development of fluoxetine-based derivatives. (A) The chemical structure of fluoxetine, N-methyl-3-[p-trifluoromethyl-phenoxy]-3-phenylpropylamine. (B) Schematic representation of the synthesis of two fluoxetine derivatives, (R)-N-ethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine (FD-2) and N-(R)-3-(trifluorophenoxy)-3-phenylpropane-imidazole (FD-4).

and FD-4 (N-(R)-3-(trifluorophenoxy)-3-phenylpropaneimidazole) were synthesized according to the scheme outlined in Fig. 1. The specific 5-HT transporter ligand fluoxetine and DA transporter ligand GBR12909 were purchased from Sigma and Tocris, respectively. Other chemicals, unless otherwise indicated, were purchased from Sigma. [2,5,6-H3]dopamine (70-80 Ci/mmol) was purchased from Amesham Pharmacia Biotech, and [I125]RTI-55 (3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester) (2,200 Ci/mmol) was purchased from Dupont-NEN.

Generation of stable transfectant cells of the human DA transporter Normal human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection, and grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (GIBCO BRL). Wild type human dopamine transporter (hDAT) cDNA, which was kindly provided by Dr. Amara of Oregon Health Sciences University, was subcloned into pcDNA3/CMV (Invitrogen) prior to transfection into HEK-293 cells by using lipofectamine (GIBCO BRL) as described elsewhere (Chae et al., 2006). Cell lines that express the human DA

New Dopamine Transporter Inhibitors

transporter stably were selected by virtue of geneticin (Invitrogen) resistance at 250 µg/mL as described previously (Galli et al., 1995). Resulting stable cell lines, named HEK-293/hDAT cells, were tested positive for GBR12909-sensitive [3H]DA uptake before maintained and expanded in the 0.025% geneticincontaining medium.

Reverse transcription-PCR Total RNA was extracted from HEK-293/hDAT cells using TRIsol (Invitrogen) according to the manufacturer's instruction. Only high purity RNA, whose absorbance ratio of 260 nm to 280 nm is higher than 1.9, was used for the cDNA synthesis. Four to 5 µg of total RNA were subjected to cDNA synthesis with oligo dT (18 base-long) and reverse transcriptase EX (Takara Shuzo). For further amplification in PCR, human DA transporter primers were designed by a primer designing software Primer3 (available at steve @genome.wi.mit.edu) to minimize a cross-contamination from other transporters. The sequence of the primers and the expected product sizes are: forward 21mer 5'-GGGAGACCCAAGCTTGGTACC-3', reverse 24mer 5'-CACTATAGAATAGGGCCCTCTAGA-3', and 1.9 kb product. All the primers were customsynthesized and purified (Bioneer). PCR was conducted with EX Taq polymerase (Takara Shuzo) according to the manufacturer's recommendation with 1 cycle of 5 min at 94oC, followed by 30 cycles of 30 sec at 94oC, 30 sec at 54oC, and 30 sec at 72oC, and 1 cycle of 1 min at 72oC. [3H]Dopamine uptake assay Uptake assays were performed as described previously (Norregaard et al., 1998; Alexander et al., 1999) with minor modification using 2,5,6,-[3H]dopamine (70-80 Ci/mmol). Transfectant HEK-293/hDAT cells were seeded in 24-well dishes (2-3 × 102 cells/well) and grown for two days before experiments. At assay, the medium was removed by aspiration, and cells were washed twice in 500 µL of uptake buffer containing (in mM): 124 NaCl. 3.25 KCl, 2 MgCl2, 2 CaCl2, 11 D-glucose, 0.2 mg/mL ascorbic acid, 0.2 pargyline, 10 HEPES, pH 7.4). The chemicals to be tested were added to the uptake buffer and the uptake was initiated by addition of [3H]dopamine in a final volume of 500 µL. After incubation for various durations at 37oC, the cells were washed three times with 500 µL of uptake buffer, lysed in 500 µL of 1.0% SDS for 30 min, and subjected to liquid scintillation counting (LS6000IC, Beckman). Nonspecific uptake, which was determined in the presence of 1 mM dopamine, was subtracted from total uptake. All determinations were performed

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in triplicate.

[125I]RTI-55 binding assay Binding assays were performed on the membrane fraction obtained from the HEK-293/hDAT transfectants with [125I]RTI-55 as described elsewhere (Boja et al., 1992). Stable transfectant cells were seeded (23 × 103 cells/plate) and grown to confluence in a 30 mm culture dish, and the membrane fractions were isolated as described below: The collected cells were lysed by incubation at 4oC for 10 min in the lysis buffer composed of 2 mM HEPES, 1 mM EDTA, pH 7.4, and subjected to centrifugation at 20,000 rpm in a Beckman JA-20 rotor for 30 min. The membrane pellets were resuspended in phosphate-buffered saline (PBS), and the protein concentration was measured by the method of Bradford (Bradford, 1976) with bovine serum albumin (BSA) as a standard. Membrane fractions (50 µg protein) in binding buffer consisting of 122 mM NaCl, 5 mM KCl, 12 mM MgCl2, 2 mg/mL glucose, 0.2 mg/mL ascorbic acid, 25 mM HEPES, pH 7.4 were mixed with 0.2 nM of [125I]RTI-55 (2,200 Ci/ mmol), and increasing amounts of test drugs were added for 30 min at room temperature. The binding was terminated by a rapid filtration over 0.2% polyethyleneimine (PEI)-pretreated GF/B glass fiber filters (Millipore), followed by 3 time-washing with the cold binding buffer in a manifold connected to a vacuum (Millipore). The radioactivity was counted in a γcounter (Gamma 5500B, Beckman). The nonspecific binding was determined in the presence of 10 µM GBR12909. FD-2 or -4 dissolved at 10 mM in 100% dimethylsulfoxide (DMSO) was subsequently diluted in binding buffer before use. Electrophysiology The HEK-293/hDAT cells were grown attached onto a cover slip and transferred into a recording chamber on the stage of an inverted microscope (IX-70, Olympus), and membrane currents were recorded using the whole-cell patch clamp technique (Hamill et al., 1981). Patch micropipettes having resistance of 3-4 MΩ were pulled by a puller (PP-83, Narishige) from borosilicate glass capillaries (TW150F-4, WPI) and fire-polished using a microforge (MF-83, Narishige). The pipette solution contained (in mM) 110 CsCl, 20 tetraethylammonium chloride (TEA-Cl), 0.24 CaCl2, 5 EGTA, 10 D-glucose, 10 HEPES to remove the K+ currents. The pH was adjusted to 7.4 with CsOH. The external solution contained (in mM) 140 NaCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, 10 HEPES. The pH was adjusted to 7.4 with NaOH. The membrane currents were recorded at 22-24oC using Axopatch 200B

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amplifier (Axon Instruments) and digitized by a 12-bit analog-to-digital interface (Digidata 1200, Axon Instruments). All of the currents were filtered at 1 or 2 KHz and sampled at 10 or 20 KHz. The bath or the dopaminergic inhibitor-containing solution was applied to the recording chamber via a gravity-fed perfusion system.

Data analysis and statistics Data were analyzed by least squares fitting with Origin4.0 (Microcal Software) on a PC. The concentration dependences of inhibition of binding or uptake (see Fig. 4) were fitted with the equation B/Bc = (IC50)nH /{(IC50)nH + [I]nH}, where Bc is bound radioligand (in binding assay) or taken up radioligand (in uptake

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assay) in the absence of inhibitor (control) and B is bound/taken up radioligand in the presence of inhibitor at [I]. IC50 is the concentration of inhibitor that decreases binding/uptake to 50% of control values, and nH is the Hill coefficient. In some cases nH was assumed to be equal to 1. Saturation isotherm of [125I]RTI-55 binding (see Fig 2 and 4) and of [3H]dopamine uptake (Fig. 3 and 4) were fitted with the general equation of Hill (Hill, 1910) B = Bm {[F]nH/([F]nH + (K50)nH)}, where B is the concentration of bound or taken up radioligand at the free concentration [F], and Bm equals the concentration of maximal binding sites or maximal amount of the taken up, and K50 is the concentration of radioligand at which half the Bm is achieved. The Eadee-Hofstee

Fig. 2. Characterization of human dopamine transporter expressions in HEK293 cells. (A) RT-PCR analysis of hDAT expressions. Stable HEK293 cell lines were generated by transfecting hDAT in pcDNA3/CMV as described in Materials and Methods. Total RNA was extracted from stable transfectant cells of hDAT, and amplified in RT-PCR with following primers: sense primer, 5'-GGGAGACCCAAGCTTGGTACC-3'; antisense primer, 5'-CACTATAGAATAGGGCCCTCTAGA-3'. The resulting cDNAs were electrophoresed on an agarose gel. Lane 1: λ DNA/HindIII DNA size markers; lane 2: cDNA from hDAT transfectants; lane 3: cDNAs from mock-transfectants. (B) Equilibrium binding of [125I]RTI-55 to the hDAT expressed in HEK293 cells. The membrane-rich fractions were obtained from the stable HEK293/hDAT cells as described in Materials and Methods. About 50 µg of the membrane proteins were incubated with increasing amount of [125I]RTI-55 in the binding buffer for 30 min. Then, the bound [125I]RTI-55 was separated from the unbound by a filtration through glass-fiber filters, and subjected to the gamma counting. The specific binding was obtained by subtracting the nonspecific binding, which was measured in a similar way except that the incubation of membranes and [125I]RTI-55 was performed in the presence of 10 µM GBR12909. The cpm values of the specific binding were transformed into moles based on the specific activity of 125I and the counting efficiency of the gamma counter. Line is from nonlinear fit with the Hill equation as described in Materials and Methods: the respective Kd and Bmax values were 0.25 nM and 0.50 pmol/mg protein. In inset, points are fitted according to the Scatchard plot.


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Fig. 3. [3H]DA uptake through the hDAT expressed in HEK293 cells. (A) Time course of [3H]DA uptake into the HEK-293/ hDAT cells. Cells were seeded at 2 × 105 cells/mL in a culture dish. 2 days after the seeding, [3H]DA was added to the dish at the final concentration of 20 nM, and incubated at 37oC with 5% CO2 for varying amounts of time from 0 to 60 min. After being washing with cold PBS several times, cells were lyzed in 1% SDS before scintillation counting. The nonspecific uptake of [3H]DA was measured similarly but in the presence of 1 mM DA. The specific [3H]DA uptake was obtained by subtracting the nonspecific uptake. Line is from nonlinear fit with the Hill equation as described in Materials and Methods. (B) Determination of [3H]DA uptake rate. The hDAT-expressing HEK293 cells were incubated with increasing concentrations of [3H]DA for 10 min to determine the [3H]DA uptake rate. The specific uptake of [3H]DA was determined as described above and represented as moles/min per 105 cells. Line is from nonlinear fits to saturation isotherm. Inset: Eadee- Hofstee plots of the data. Line is from linear fit with B = -K50 (B/[F]) + Bm as described in Materials and Methods: Vmax = 269.8 pmol [3H]DA/ min/106 cells and Km = 34.10 µM.

plot was fitted to the following equation: B = -K50 (B/ [F]) + Bm Data are mean ± S.E.M. values. Statistical significance was evaluated by Student's t test.

RESULTS AND DISCUSSION Stable expression of hDAT in HEK-293 cells A stable cell line heterologously expressing hDAT was established by the transfection of HEK-293 cells with hDAT cDNA and selection via geneticin resistance, being referred to as HEK-293/hDAT cells. Confirmation of the DA transporter expression in HEK293/hDAT cells was conducted in two ways. First, the hDAT mRNA expression was examined. As illustrated in Fig. 2A, a RT-PCR analysis revealed a unique cDNA band in HEK-293/hDAT (lane 2) that is absent from mock-transfectants (lane 3). The size of the cDNA band (1.9 Kb) is consistent with the predicted value based on primers used. Secondly, the hDAT protein expression was examined by use of radioligand binding assay with [125I]RTI-55, which was also known as an antagonist for DAT (Boja et al., 1992). Membranes prepared from HEK-293/hDAT cells, but not from mock-transfectants, displayed saturable binding of [125I]RTI-55 with a Kd = 0.25 nM and a Bmax = 0.50 pmol/mg protein of membrane fraction (Fig. 2B). The values were comparable with those from a previous

study, where human serotonin transporters stably expressed in HEK-293 cells were probed with [125I] RTI-55, generating a Kd = 0.26 nM and a Bmax = 1.2 pmol/mg membrane protein (Qian et al., 1997). Based on protein yield per cell, an average value of ~2.5 × 104 [125I]RTI-55 binding sites per cell was estimated.

[3H]dopamine uptake mediated by hDAT To investigate characteristics of dopamine uptake in HEK-293/hDAT cells, the time-dependent and concentration-dependent uptake of [3H]DA was measured. Fig. 3A showed the time-dependent uptake of [3H]DA in the physiological saline containing 124 mM NaCl. The uptake was linear up until about 10 min after the onset, and progressively reduced until reaching the plateau at about 20 min. In Fig. 3B, the rate of [3H] DA uptake was found to be dependent on the concentration of [3H]DA. [3H]DA transport was saturable and followed the hyperbolic relation described by Michaelis-Menten kinetics with a Km = 34.10 µM and a Vmax = 269.8 pmol DA/min/106 cells. Assuming all the hDATs are available for transport, it is estimated that the turnover rate of hDAT, which is equal to Vmax/Bmax, is ~1.08 × 102 sec-1. Inhibitory effects of fluoxetine analogs on the [3H]dopamine uptake The inhibitory effect of fluoxetine analogs on hDAT

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was then examined by measuring the uptake of [3H]DA into HEK-293/hDAT cells in the presence of fluoxetine analogs. Under the physiologically relevant salt condition, the uptake of [3H]DA into HEK-293/

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hDAT cells was inhibited by compounds known to inhibit the DA transporter in various tissue preparations, including GBR12909 (Andersen, 1989; Tatsumi et al., 1997) and phorbol 12-myristate 13-acetate (β-

Fig. 4. Effects of various inhibitors on [3H]DA uptake in the hDAT-expressing HEK293 cells. Cells were seeded at 2 × 105 cells/mL in a culture dish. 2 days after the seeding, cells were pretreated with either GBR12909 (A), a well-known hDAT inhibitor, PMA (B), a PKC activator, fluoxetine (C), FD-2 (D), or FD-4 (E) at the indicated concentration for 30 min before [3H]DA was added at the final concentration of 20 nM. Cells were then incubated in the mixture further for 10 min before washing. Lysis and scintillation counting were performed as described in Materials and Methods. The line is from nonlinear fits to the equation B/Bc = (IC50)nH/{(IC50)nH + [I]nH}. The IC50 values of GBR12909, PMA, fluoxetine, FD-2 and FD-4 are 0.34 ± 0.011 µM (n = 3), 0.028 ± 0.0051 mM (n = 3), 0.21 ± 0.032 mM (n = 3), 0.077 ± 0.0032 mM (n = 3) and 0.26 ± 0.13 mM (n = 3), respectively


PMA), protein kinase C activator (Qian et al., 1997) in a concentration-dependent manner (Fig. 4A and 4B) with IC50 values of 0.34 ± 0.011 µM (n = 3) and 28.1 ± 1.2 µM (n = 3), respectively. Fluoxetine also inhibited the [3H]DA uptake with IC50 = 205 ± 32 µM (n = 3) (Fig. 4C). Similarly, two fluoxetine analogs, FD-2 and FD-4, inhibited the [3H]DA uptake with IC50 = 77.0 ± 3.2 µM (n = 3) and 25.5 ± 1.3 µM (n = 3), respectively (Fig. 4D and 4E), indicating that FD-2 and FD-4 are more potent than fluoxetine in inhibiting the [3H]DA uptake by as much as 3-4 folds. Consistently, both FD-2 and FD-4 exhibited the potency for inhibiting [125I]RTI-55 binding to hDAT (data not shown).

FD-2-sensitive, DA transporter-associated currents The hDAT-mediated inward currents were elicited by DA application in a voltage-clamped hDAT-expressing oocyte (Sonders et al., 1997; Zhu et al., 1997). Similarly, the whole-cell configuration of the patchclamp technique was applied to investigate the DA-

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induced ionic currents in HEK-293/hDAT cells. In these experiments, the cells were bathed in normal HEPES-buffered saline solution containing 2.0 mM CaCl2 and impaled with a pipette solution containing 110 mM CsCl and 20 mM tetraethylammonium chloride (TEA-Cl) to remove K+ currents. Experiments were performed with FD-2 which is about 3-4 times more potent than FD-4 in inhibiting dopamine transporter (Fig. 4D and Fig. 4E). As shown in Fig. 5, the membrane potential was at -60 mV (holding potential), and voltage pulses ranging from -120 to +20 mV at a 20-mV increment and a duration of 400 msec (test potential) were applied. The test pulses elicited inward and outward currents in the presence of 1 µM DA (Fig. 5A) or in the presence of 1 µM DA plus 10 µM FD-2 (Fig. 5B). Outward currents, which were elicited above -60 mV, were likely to be independent of FD-2, whereas the inward currents were decreased in amplitude in the presence of 10 µM FD-2. Therefore, the FD-2-sensitive, thus seemingly transporter-mediated currents (Fig. 5C) were obtained by subtracting the

Fig. 5. Effects of FD-2 on the DA transporter-associated currents. The HEK-293/hDAT cells were bathed in HEPESbuffered saline as described in Materials and Methods and held at -60 mV, and command pulses were applied from -120 to +20 mV in 20-mV increment at a duration of 400 msec. Representative current traces were elicited by applying 1 µM DA alone (A) or by co-applying DA and FD-2 at 1 µM each (B). The current traces in (C) were generated by subtracting those in (B) from those in (A).

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current traces shown in Fig. 5B from those in Fig. 5A. Although the size of the current is small and thus noisy, the resulting FD-2-sensitive inward currents represent hDAT-mediated transport currents in many respects. First, the current is inward consistently with Sonders et al. (1997). At -120 mV, the whole-cell current induced by 1 µM DA was typically ~30 pA, which is comparable to the value obtained from 5-HT transporter (Hamill et al., 1981). Secondly, by substitution of Cs+ for Na+ and addition of TEA in the pipette solution to completely block any K+ current, Na+/Cl−-coupled DA transporter-associated current was isolated. This hDAT-associated current was shown to be sensitive to FD-2. Although the test compounds are derived from the fluoxetine, selective serotonin uptake inhibitor, their effects on the serotonin transporter or other amine transporters were not examined in this study. For now, their actions on the dopamine transporter were solely tested and then their pharmacological properties will be further characterized in detail in separate experiments. Taken together, the two N-methyl amine derivatives of fluoxetine, FD-2 and FD-4 act as a new class of dopamine transporter inhibitors with high affinity toward the human dopamine transporter. They are effective in (i) inhibiting [3H]DA uptake via hDAT, (ii) inhibiting [125I]RTI-55 binding to hDAT, and (iii) blocking the DA-induced currents mediated by hDAT, at least, in HEK-293 cells. Especially, FD-2 is about 34 times more potent than FD-4 in inhibiting dopamine transporter. The dopamine transporter (DAT) has been suggested to be the critical recognition site for cocaine, thereby mediating its abuse liability. Discovery of dopamine transporter inhibitors would be useful with regard to substituting for cocaine and minimizing its abuse. Therefore, these newly synthesized fluoxetine derivatives may be useful as a new class of dopamine transporter inhibitors for substituting cocaine.

ACKNOWLEDGEMENTS The hDAT cDNA was kindly provided by Dr. Susan Amara of Oregon Health Sciences University, OR, and this work was supported by the grant KRF-2007-313C00733 from Korea Research Foundation to C. Min.

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