Studies of the Biogenic Amine Transporters. 12. Identification of Novel Partial Inhibitors of Amphetamine-Induced Dopamine Release

The biogenic amine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), are important targets for a wide range of medications used to treat a variety of psychiatric conditions, such as anxiety, depression, and obsessive compulsive disorder (Gorman and Kent, 1999; Zohar and Westenberg, 2000). Drugs that interact with transporters generally interact with these proteins in two distinct ways. Reuptake inhibitors bind to transporter proteins but are not transported. These drugs elevate extracellular concentrations of transmitter by blocking transporter-mediated uptake of transmitters from the synapse. Substrate-type releasers bind to transporter proteins and are subsequently transported into the cytoplasm of nerve terminals, releasing neurotransmitter via a process of carrier-mediated exchange (Rudnick and Clark, 1993; Rothman and Baumann, 2006).

There is growing interest in the possible therapeutic potential of allosteric modulators (Christopoulos and Kenakin, 2002; Schwartz and Holst, 2007), including the identification of allosteric modulators of the biogenic amine transporters (BATs) (Sanchez, 2006). Early evidence of allosteric interactions at the biogenic amine transporters included our finding that pretreatment of guinea pig membranes with paroxetine increased the dissociation rate of [³H]cocaine from SERT (Akunne et al., 1992). Using rat SERT expressed in HEK cells, Sur et al. (1998) presented evidence that imipramine allosterically modulated the ability of citalopram to inhibit [³H]5-hydroxytryptamine transport. Others reported apparent allosteric interactions between 5-hydroxytryptamine and [³H]paroxetine binding to human platelet SERT (Andersson and Marcusson, 1989) and between β-estradiol and SERT (Chang and Chang, 1999). More recently, we reported novel allosteric modulators of both DAT (SoRI-9804) (Rothman et al., 2002) and SERT (SoRI-6238 and TB-1-099) (Nandi et al., 2004; Nightingale et al., 2005). Moreover, Chen et al. (2005) reported evidence for allosteric modulation of [³H]S-citalopram binding (Chen et al., 2005).

In 1999, we searched a library of compounds maintained by Southern Research Institute (Birmingham, AL) for compounds that possessed a diphenylmethyl (benzhydryl) group. Using rat brain tissue assays, we screened these compounds for activity in binding assays for DAT, SERT, and NET (unpublished data). This effort identified several possible allosteric modulators of the BATs. We examined in greater detail the interaction of selected agents with the BATs. SoRI-9804 (Fig. 1) partially inhibited [¹²⁵I]RTI-55 binding to DAT and partially inhibited [³H]DA uptake by rat brain synaptosomes. SoRI-6238, and a subsequent compound that was not part of the SoRI library (TB-1-099), were shown to allosterically modulate SERT (Nandi et al., 2004; Nightingale et al., 2005). In the present study, we focused on three additional compounds identified as being potential allosteric modulators (Fig. 1): SoRI-20040, SoRI-20041, and SoRI-2827. Initial screens indicated that all three agents were inactive at NET and SERT binding (IC₅₀ > 10 μM) but inhibited [¹²⁵I]RTI-55 binding to the rat brain DAT in a manner suggestive of allosteric interactions. We report here that these three agents allosterically modulate the human DAT (hDAT) expressed in HEK cells and noncompetitively inhibit [³H]DA uptake by rat caudate synaptosomes.

Materials and Methods

Animals. Male Sprague-Dawley rats (300–450 g), used for ³H neurotransmitter uptake assays, were obtained from Charles River Laboratories, Inc. (Wilmington, MA). The animal housing facilities were fully accredited by the American Association of the Accreditation of Laboratory Animal Care, and all experiments were performed within the guidelines delineated in the Institutional Care and Use Committee of the National Institute on Drug Abuse, Intramural Research Program.

Tissue Preparation. HEK cells expressing hDAT were grown to confluence on plates, using published methods (Nightingale et al., 2005). The medium was removed, and the plates were stored at –80°C until the day of the assay. The plates were thawed, and then the cells were scraped off and rinsed with 55.2 mM sodium phosphate buffer, pH 7.4 (BB), and homogenized with a Polytron homogenizer at setting 6 for 10 s. The homogenate was centrifuged twice at 30,000g for 10 min, with resuspensions in BB. The final pellets were resuspended in a final volume of 225 ml/plate with BB.

Transporter Binding Assays. For assays using HEK cells expressing hDAT, experiments were carried out in 12- × 75-mm polystyrene tubes that were prefilled with 100 μl of drug, 100 μl of radioligand, and 50 μl of a blocker or buffer (Rothman et al., 1998). [¹²⁵I]RTI-55 was diluted in a protease inhibitor cocktail (BB with 25 μg/ml chymostatin, 25 μg/ml leupeptin, 1 mM EGTA, and 1 mM EDTA). The drugs and blockers were made up in BB with 1 mg/ml bovine serum albumin at pH 7.4. The experiment was initiated with the addition of 750 μl of membranes in BB. Samples were incubated in a final volume of 1 ml, at 0°C for 18 to 24 h (steady state). After incubation, the samples were filtered with a cell harvester (Brandel Inc., Gaithersburg, MD), over Whatman GF/B filters (Whatman, Clifton, NJ) presoaked in wash buffer (ice-cold 10 mM Tris-HCl and 150 mM NaCl, pH 7.4) containing 2% poly(ethyleneimine). Typical total and nonspecific cpms observed for the HEK cell transporter binding assays were 2000 and 75, respectively.

Kinetic Experiments. Kinetic experiments were conducted with minor modifications of published methods (Nandi et al., 2004). For hDAT dissociation experiments, [¹²⁵I]RTI-55 (0.01 nM) was incubated for 2 h at 25°C (steady state). At that point, 100 μl of RTI-55 (final concentration 1 μM) or buffer was added. Ten minutes later, test drug (10 μM) was added. This design ensured that any effect of the test drug could not be due to interactions with the DAT binding site, because this site would be completely occupied by RTI-55. Triplicate samples were filtered at various times after the addition of test drug: 5, 15, 25, 35, and 55 min. For the data analyses (described below), 100% of control point was time 0 min for conditions that did not receive test drug and time 10 min for conditions that received test drug.

Neurotransmitter Uptake Assays. [³H]DA uptake inhibition assays were conducted as described previously (Rothman et al., 2001). Freshly removed caudate ([³H]DA uptake) was homogenized in 10% ice-cold sucrose with 12 strokes of a hand-held Potter-Elvehjem homogenizer followed by centrifugation at 1000g for 10 min. The supernatants were saved on ice and used immediately. The assay buffer used was Krebs' phosphate buffer containing 154.4 mM NaCl, 2.9 mM KCl, 1.1 mM CaCl₂, 0.83 mM MgCl₂, 5 mM glucose, 1 mg/ml ascorbic acid, and 50 μM pargyline. Nonspecific uptake was measured by incubating in the presence of 10 μM indatraline. The reactions were stopped after 15 min by filtering with a cell harvester (Brandel Inc.) over Whatman GF/B filters (Whatman) presoaked in wash buffer maintained at 25°C (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl). Retained tritium was measured with a Trilux liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Shelton, CT) after overnight extraction in 0.6 ml of liquid scintillation cocktail (MP Biomedicals, Solon, OH). Typical total and nonspecific cpms observed for the rat brain transporter uptake inhibition assays were 18,000 and 1000, respectively.

[³H]MPP⁺Release Assays. This assay measures the ability of test agents to release preloaded [³H]MPP⁺ via the DAT using rat striatal synaptosomes. The details of this assay were published previously (Rothman et al., 2003).

Experimental Design. Inhibition curves were generated by displacing one to three concentrations of [¹²⁵I]RTI-55 by 10 concentrations of drug. Concentrations of [¹²⁵I]RTI-55 greater than 0.01 nM were generated by addition of nonradioactive RTI-55. For binding surface experiments (Rothman, 1986; Rothman et al., 1991), two different concentrations of radioligand were each displaced by 10 concentrations of test agents in the absence or presence of various blockers. Surfaces for [³H]DA uptake were generated by displacing two concentrations of [³H]DA (∼2 and 20 nM) by DA (2–4096 nM) in the absence and presence of three concentrations of test agent. The higher concentration of [³H]DA was obtained by adding DA to the radioligand. Dissociation experiments were conducted with minor modification of published procedures (Rothman et al., 1991).

Data Analysis and Statistics. Inhibition curve data were expressed as “percentage of inhibition” and fit to the following two-parameter dose-response curve model: Y = E_max × ([D]/([D] + EC₅₀) for the best fit estimates of the E_max and EC₅₀ values using either

KaleidaGraph version 3.6.4 (Synergy Software, Reading, PA) or MLAB-PC (Nightingale et al., 2005). Binding surfaces were fit to one- and two-site binding models using MLAB-PC as described previously (Rothman et al., 1991). Dissociation experiments were fit to one- or two-component dissociation models (Nandi et al., 2004). Statistical significance among binding models was determined using the F-test, with a threshold of p < 0.01. Graphs were generated with KaleidaGraph 3.6 software.

Chemicals. [³H]DA (specific activity = 31.8 Ci/mmol) was from PerkinElmer Life and Analytical Sciences. The sources of other chemicals were published previously (Rothman et al., 1998).

Results

The first series of experiments determined the inhibitory effect of SoRI-20040, SoRI-20041, and SoRI-2827, in comparison with cocaine, on [¹²⁵I]RTI-55 binding to hDAT. As reported in Fig. 2A, cocaine inhibited [¹²⁵I]RTI-55 binding in a classic dose-dependent manner, and the dose-response curve shifted to the right as the concentration of [¹²⁵I]RTI-55 increased from 0.01 to 0.1 and 1 nM. The extrapolated E_max values were ∼100% for all three concentrations of [¹²⁵I]RTI-55. In contrast, the inhibition curves observed for the SoRI compounds were atypical (Table 1; Figs. 2 and 3). For example, all three agents partially inhibited [¹²⁵I]RTI-55 binding, with E_max values decreasing as the concentration of [¹²⁵I]RTI-55 increased. Using 1.0 nM [¹²⁵I]RTI-55, we observed the following E_max values: SoRI-20040 (62%), SoRI-20041 (44%), and SoRI-2827 (39%). In addition, as reported in Table 1, the EC₅₀ values of SoRI-2827 (Fig. 3B) did not significantly shift to the right as observed with cocaine.

The second series of experiments determined the effect of three concentrations of test agent on the K_d and B_max values of the hDAT as measured with [¹²⁵I]RTI-55. It is expected that increasing the concentration of a competitive inhibitor would have no effect on the B_max value but would increase the apparent K_d value linearly according to the equation K_d(apparent) = K_d × (1 + [I]/K_I). As reported in Fig. 4A, 0.8 and 12.8 μM SoRI-20040 significantly reduced the B_max value by 17 and 32%, respectively. SoRI-20040 also increased the apparent K_d value in a dose-dependent manner that was well described by a sigmoidal dose-response curve, rather than the linear curve expected of a competitive inhibitor. SoRI-20041 (Fig. 5) significantly decreased the B_max value at all three test concentrations and also increased the apparent K_d value in a dose-dependent manner, which was also well described by a sigmoidal dose-response curve. Similar results were observed for SoRI-2827, except that this compound decreased the B_max value only at the 3.2 μM concentration (Fig. 6).

The next series of experiments determined the effect of the SoRI agents on the dissociation kinetics of [¹²⁵I]RTI-55 binding to hDAT. Table 2 reports the experimental design used for these experiments. In brief, addition of RTI-55 (1 μM) initiated the dissociation of [¹²⁵I]RTI-55 binding, after which test drugs were added so that any effect of the test agent could not be due to an effect at the transporter binding site, because these were prebound with RTI-55. As reported in Fig. 7A and Table 3, the dissociation of [¹²⁵I]RTI-55 from hDAT initiated by 1 μM RTI-55 proceeded in a monotonic manner and was well described by a single component dissociation model (K_–1 = 0.055 ± 0.002 min^–1). The addition of SoRI-20040 after the addition of RTI-55 significantly slowed the dissociation rate (K_–1 = 0.038 ± min^–1 min^–1). The dissociation of [¹²⁵I]RTI-55 initiated by the addition of SoRI-20040 alone was well described by a two-component dissociation model. The dissociation rate of the A1 component (K_–2 = 0.059 ± 0.029) was similar the dissociation rate observed in the RTI-55 condition. The dissociation rate of the A2 component (K_–1 = 0.0047 ± 0.013) was quite slow and had a large S.D. A similar pattern of results was observed for SoRI-20041, except that the addition of SoRI-20041 alone resulted in a zero dissociation rate for the A2 component reflecting the trend for an increased level of binding at the 60-min point (Fig. 7B). Unlike SoRI-20040 and SoRI-20041, SoRI-2827 did not influence the dissociation rate of [¹²⁵I]RTI-55 from hDAT (Fig. 7C; Table 3).

We next determined the effect of the SoRI compounds on [³H]DA uptake by rat caudate synaptosomes. As reported in Table 4, the SoRI compounds partially inhibited [³H]DA uptake, with E_max values ranging from 68 to 78%, and EC₅₀ values ranging from 1770 to 3120 nM. In contrast, indatraline and GBR12909 did not partially inhibit [³H]DA uptake. All three SoRI compounds inhibited [³H]DA uptake in a manner inconsistent with competitive inhibition (Fig. 8; Table 5). SoRI-20040 increased the apparent K_M value by ∼35% at all three doses tested and decreased the V_max value in a dose-dependent manner. As reported in Fig. 8B, the EC₅₀ value for SoRI-20040 decreasing the V_max value was 1.7 μM, with an E_max value of 90%. Similar results were observed for SoRI-20041, except that the lowest concentration of SoRI-20041 tested (0.8 μM) had already decreased the V_max value by 70%. The estimated EC₅₀ value for SoRI-20041 was 0.16 μM. SoRI-2827, in contrast to the other agents, was less potent at decreasing the V_max value.

Cocaine and indatraline are competitive inhibitors of [³H]DA uptake. Using indatraline as a control drug, we assessed the effect of the SoRI compounds on cocaine-induced inhibition of [³H]DA uptake. As reported in Table 6, 7 nM indatraline reduced specific [³H]DA uptake to 31% of control and increased the IC₅₀ value of cocaine from 278 to 894 nM. SoRI-20040 (12.8 μM) reduced specific [³H]DA uptake to 43% of control and produced a much smaller increase in the IC₅₀ value of cocaine (450 nM) than did indatraline. Similar results were obtained with SoRI-20041 and SoRI-2827.

In light of the profound effect of the SoRI compounds on the V_max value of [³H]DA uptake (Table 5), we determined the effect of these agents on d-amphetamine-induced release of [³H]MPP⁺ from striatal dopamine nerve terminals (Rothman and Baumann, 2006). As reported in Fig. 9 and Table 7, d-amphetamine produced a dose-dependent release of [³H]MPP⁺, with an EC₅₀ value of ∼6 nM and an E_max value of 100%. Cocaine (2.8 μM) shifted the d-amphetamine curve to the right (EC₅₀ = 82 nM) without altering the E_max value. SoRI-9804 produced a dose-dependent decrease in the E_max value, resulting in an E_max value of 73% with the 10 μM dose. Similar results were obtained with SoRI-20040 (Fig. 10; Table 7). In these experiments, indatraline (66 nM) shifted the d-amphetamine curve to the right (EC₅₀ = 116 nM) without altering the E_max value. SoRI-20040 produced a dose-dependent decrease in the E_max value, resulting in an E_max value of 60% with the 10 μM dose. At 10 μM, SoRI-9804 did not affect [³H]MPP⁺ release and SoRI-20040 had a minimal effect (∼15%). The effects of SoRI-20041 and SoRI-2827 on d-amphetamine-induced release of [³H]MPP⁺ are more complex than observed for SoRI-9804 and SoRI-20040 (data not shown) and will be published in due course.

Discussion

Based on our experience screening hundreds of compounds in biogenic amine transporter binding assays (Prisinzano et al., 2004), most agents inhibited rat brain DAT binding with slope factors (Hill coefficients) close to 1, a finding strongly suggestive of a binding process governed by simple mass action kinetics. SoRI-9804 was the first compound we came across that inhibited [¹²⁵I]RTI-55 binding to DAT in a qualitatively different manner (Rothman et al., 2002). As observed in this study with SoRI-20040, SoRI-20041, and SoRI-2827, SoRI-9804 partially inhibited DAT binding and [³H]DA uptake. We reported previously on other compounds that allosterically modulate SERT (Nandi et al., 2004; Nightingale et al., 2005).

Early hints of allosteric interactions at the biogenic amine transporters included our finding that pretreatment of guinea pig membranes with paroxetine increased the dissociation rate of [³H]cocaine from SERT (Akunne et al., 1992). Using rat SERT expressed in HEK cells, Sur et al. (1998) presented evidence that imipramine allosterically modulated the ability of citalopram to inhibit [³H]5-HT transport. Others reported apparent allosteric interactions between 5-HT and [³H]paroxetine binding to human platelet SERT (Andersson and Marcusson, 1989) and between β-estradiol and SERT (Chang and Chang, 1999). More recently, we reported novel allosteric modulators of both DAT (Rothman et al., 2002) and SERT (Nandi et al., 2004), and Chen et al. (2005) reported evidence for allosteric modulation of [³H]S-citalopram binding.

Several lines of evidence support the hypothesis that SoRI-20040, SoRI-20041, and SoRI-2827 allosterically modulate hDAT. First, all three agents partially inhibit [¹²⁵I]RTI-55 binding to hDAT. The partial inhibition is most apparent with the higher concentrations of [¹²⁵I]RTI-55. In this case, the E_max values range from 39% inhibition for SoRI-2827 to 62% for SoRI-20040. We observed similar results for the partial inhibition of μ opioid receptor binding by salvinorin A (Rothman et al., 2007). These observations suggest that the fractional occupation of the receptor by the radioligand affects the ability of the allosteric drug to displace binding. Second, all three SoRI compounds affected the K_d and B_max values of [¹²⁵I]RTI-55 binding to hDAT in a manner unlike that of a competitive inhibitor. All three agents decreased the B_max and increased the apparent K_d value in a dose-dependent manner (Figs. 3, 4, 5, 6) (mixed inhibition model). The dose-response curves were well described by a sigmoidal dose-response curve, rather than the linear curve expected of a competitive inhibitor. The low micromolar EC₅₀ values for increasing the apparent K_d values (Figs. 4, 5, 6) were similar to the EC₅₀ values for inhibition of [¹²⁵I]RTI-55 binding to hDAT (Table 1).

The dissociation experiments provide a third line of evidence indicating that the SoRI-20040 and SoRI20041 allosterically modulate hDAT (Fig. 7). Dissociation experiments are classically used to detect allosteric effects, specifically comparing the dissociation rate observed when dissociation is initiated by dilution versus the addition of an excess of a competing ligand. However, as described in our previous work (Nandi et al., 2004), the dilution method does not work in the hDAT binding assay, because reassociation of the radioligand occurs during the dissociation experiment. Thus, we determined the ability of a test agent to alter [¹²⁵I]RTI-55 dissociation initiated by the addition of 1 μM RTI-55. The test agents were added 10 min after the RTI-55, ensuring that any effect observed could not be due to interactions at the RTI-55 hDAT binding site (Table 2). As reported in Table 3, SoRI-20040 and SoRI-20041, but not SoRI-2827, slowed the dissociation rate, providing clear evidence of an allosteric effect. The addition of the SoRI compounds in the absence of RTI-55 resulted in biphasic dissociation curves, the faster component of which (A1) was similar to that observed with the addition of RTI-55. Because these compounds do not completely inhibit [¹²⁵I]RTI-55 binding, the simplest explanation of the biphasic dissociation curves is that these compounds initiated a more rapid dissociation (K_–1) followed by a much slower (K_–2) dissociation and a reassociation of [¹²⁵I]RTI-55 binding.

The [³H]DA uptake experiments provide the fourth line of evidence that SoRI-20040, SoRI-20041, and perhaps SoRI-2828 are allosteric modulators of DAT. All three compounds increased the apparent K_M values in a nonlinear manner. For example, SoRI-20040 increased the K_M value from 36.8 to ∼50 nM at all three concentrations tested (0.8, 3.2, and 12.8 μM). Only one compound (SoRI-20041) increased the K_M value in a dose-dependent manner. In this case, the data were well described by a sigmoidal dose-response curve, with an EC₅₀ value of 1.6 ± 0.02 μM and an E_max value of 73 ± 0.2% (data not shown). All three SoRI compounds decreased the V_max value of [³ H]DA uptake. SoRI-20040 and SoRI-20041 decreased the V_max value in a dose-dependent manner (Fig. 8). Note that, with an EC₅₀ value of 0.16 μM, SoRI-20041 was especially potent at decreasing the V_max value.

Finally, the experiments reported in Figs. 9 and 10 and Table 7 strongly suggest that SoRI-9804 and SoRI-20040 do not interact with DAT in a competitive manner. Cocaine and indatraline, competitive inhibitors at DAT, shifted the d-amphetamine dose-response curve for releasing preloaded [³H]MPP⁺ via DAT to the right, without altering the E_max value. In contrast, 10 μM SoRI-9804 (Fig. 9) and 10 μM SoRI-20040 (Fig. 10), which by themselves had minimal effects on [³H]MPP⁺ release, substantially decreased the E_max value for the d-amphetamine dose-response curve, with minimal changes in the EC₅₀ value. These data indicate that SoRI-9804 and SoRI-20040 act as partial inhibitors of d-amphetamine-induced DAT-mediated [³H]MPP⁺ release. Similar data were obtained using [³H]DA instead of [³H]MPP⁺ (data not shown). To our knowledge, such partial inhibitors of DAT-mediated release have not been reported.

In summary, various studies over the years hint of an allosteric binding site associated with the biogenic amine transporters. The data presented here indicate that SoRI-20040 and SoRI-20041 allosterically modulate DAT and that SoRI-2827 interacts with DAT in a manner not consistent with that of simple competitive inhibitor. Our study therefore presents strong evidence for such an allosteric modulatory site and identifies novel partial inhibitors of DAT-mediated dopamine release. Such partial inhibitors, by reducing the effects of amphetamine, might be of use in the treatment of methamphetamine dependence.