QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.084376v1 314/2/906    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nightingale, B. Articles by Rothman, R. B. Search for Related Content PubMed PubMed Citation Articles by Nightingale, B. Articles by Rothman, R. B.

NEUROPHARMACOLOGY

Studies of the Biogenic Amine Transporters. XI. Identification of a 1-[2-[Bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) Analog That Allosterically Modulates the Serotonin Transporter

Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland (B.N., C.M.D., R.B.R.); and Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland (T.L.B., E.G., W.J.C., A.E.J., K.C.R.)

Received February 1, 2005; accepted April 20, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Previous studies identified partial inhibitors of serotonin (5-HT) transporter and dopamine transporter binding. We report here on a partial inhibitor of 5-HT transporter (SERT) binding identified among a group of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine analogs (4-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-1-(2-trifluoromethyl-benzyl)-piperidine; TB-1-099). Membranes were prepared from rat brains or human embryonic kidney cells expressing the cloned human dopamine (hDAT), serotonin (hSERT), and norepinephrine (hNET) transporters. -(4'-¹²⁵Iodophenyl)tropan-2-carboxylic acid methyl ester ([¹²⁵I]RTI-55) binding and other assays followed published procedures. Using rat brain membranes, TB-1-099 weakly inhibited DAT binding (K_i = 439 nM), was inactive at NET binding ([³H]nisoxetine), and partially inhibited SERT binding with an extrapolated plateau ("A" value) of 20%. Similarly, TB-1-099 partially inhibited [¹²⁵I]RTI-55 binding to hSERT with an extrapolated plateau (A value) of 14%. Upon examining the effect of increasing concentrations of TB-1-099 on the apparent K_d and B_max of [¹²⁵I]RTI-55 binding to hSERT, we found that TB-1-099 decreased the B_max in a dose-dependent manner and affected the apparent K_d in a manner well described by a sigmoid dose-response curve. TB-1-099 increased the K_d but not to the magnitude expected for a competitive inhibitor. In rat brain synaptosomes, TB-1-099 noncompetitively inhibited [³H]5-HT, but not [³H]dopamine, uptake. Dissociation experiments indicated that TB-1-099 promoted the rapid dissociation of a small component of [¹²⁵I]RTI-55 binding to hSERT. Association experiments demonstrated that TB-1-099 slowed [¹²⁵I]RTI-55 binding to hSERT in a manner unlike that of the competitive inhibitor indatraline. Viewed collectively, these results support the hypothesis that TB-1-099 allosterically modulates hSERT binding and function.

Drugs that interact with transporters generally interact with this protein 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. Releasers elevate extracellular transmitter concentrations by a two-pronged mechanism: 1) they increase cytoplasmic levels of transmitter by disrupting storage of transmitters in vesicles, and 2) they promote non-exocytotic release of transmitters by a process of carrier-mediated exchange (Rudnick and Clark, 1993). Although most therapeutic agents that target the SERT inhibit its function, an effect termed uptake inhibition, there is emerging evidence that SERT substrates may offer unique therapeutic effects (Rothman and Baumann, 2002a,b).

Several different types of assays can be used to characterize the interaction of test agents with the biogenic amine transporters. These include nonfunctional ligand binding assays, where compounds are tested for their ability to inhibit the binding of a radioactive transporter inhibitor such as 3-(4'-¹²⁵iodophenyl)tropan-2-carboxylic acid methyl ester ([¹²⁵I]RTI-55) to the transporter and functional assays where compounds are tested for their ability to inhibit the reuptake of ³H-neurotransmitter by synaptosomes. For the sake of brevity, we refer in this study to the transporter ligand binding assays, which measure the affinity of a test compound for the transporter inhibitor site of the transporter, as SERT, NET (norepinephrine transporter), and DAT (dopamine transporter) binding assays. Similarly, we refer to the uptake inhibition assays as either SERT, NET, and DAT uptake assays or [³H]5-HT, [³H]NE, or [³H]dopamine (DA) uptake assays.

As part of our ongoing studies of the biogenic amine transporters and efforts to develop novel agents to test the DA hypothesis of cocaine addiction, we have characterized hundreds of compounds at SERT using both ligand binding assays and functional assays (Prisinzano et al., 2004). Until recently, active compounds inhibited [¹²⁵I]RTI-55 binding to the SERT in a manner consistent with mass action. Recently, however, we reported an agent, SoRI-6238, that partially inhibited SERT binding, allosterically modulated the dissociation rate of [¹²⁵I]RTI-55 binding from rat brain SERT, and allosterically modulated SERT function (Nandi et al., 2004), as indicated by noncompetitive inhibition of the [³H]5-HT uptake. Previously, we reported a novel compound, SoRI-9804 (4-[(diphenylmethyl)amino]-2-phenylquinazoline), that partially inhibited [¹²⁵I]RTI-55 binding to the DAT and [³H]DA uptake by rat brain synaptosomes (Rothman et al., 2002). In the present report we describe a GBR12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine) analog, TB-1-099 (4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-1-(2-trifluoromethyl-benzyl)-piperidine), that appears to allosterically modulate certain properties of hSERT expressed in HEK cells, such as SERT binding and SERT uptake.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

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

Tissue Preparation. The transporter binding assays used membranes prepared from frozen rat brains (Nandi et al., 2004). Briefly, frozen rat brains (Pel Freeze, Rogers, AZ) were each thawed in 20 ml of ice-cold binding buffer (BB; 55.2 mM sodium phosphate buffer, pH 7.4) for 15 min and then homogenized with a Brinkmann Polytron (Brinkmann Instruments, Westbury, NY; setting 6 for 20 s). The homogenates were centrifuged twice at 20,000g for 15 min, with resuspensions in equal volumes of ice-cold BB, and then resuspended in 10 ml/brain ice-cold binding buffer. The membranes were pooled, mixed, and then separated into 1-ml aliquots in polypropylene microcentrifuge tubes. Each aliquot was centrifuged in a TOMY refrigerated microcentrifuge (model MTX 150; Tomy Seiko, Tokyo, Japan) at maximum speed for 5 min; the supernatant was discarded. The aliquots were then stored at -80°C until needed. When used in the experiment, each aliquot was diluted in 120 ml of binding buffer.

HEK cells expressing hNET, hDAT, or hSERT were grown to confluency using standard methods on plates, medium was removed, and the plates were stored at -80°C until the day of the assay. The plates were thawed, and the cells scraped off and rinsed with BB and homogenized with a polytron at setting 6 for 10 s. The homogenate was centrifuged twice at 30,000g for 10 min, with resuspensions in phosphate buffer. The final pellets were resuspended in a final volume of 300 ml/plate with BB.

Transporter Binding Assays. For rat brain transporter binding assays, experiments were carried out in 12 x 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). For assay of the SERT and DAT, the blockers were either 100 nM 4-(2-benzhydryloxy-ethyl)-1-(4-nitro-benzyl)piperidine oxalate to block the DAT or 100 nM citalopram to block SERT. [¹²⁵I]RTI-55 was made in a protease inhibitor cocktail (BB with 25 µg/ml chymostatin, 25 µg/ml leupeptin, 1 µM EGTA, and 1 µM 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 4°C for 18 to 24 h (steady state). After incubation the samples were filtered with a Brandel cell harvester (Brandel, Gaithersburg, MD) over Whatman GF/B filters (Brandel) presoaked in wash buffer (ice-cold 10 mM Tris/HCl, and 150 mM NaCl, pH 7.4) containing 2% poly(ethyleneimine). For assay of the rat NE transporter (NET), we used [³H]nisoxetine (American Radiolabeled Chemicals, St. Louis, MO) as described elsewhere (Rothman et al., 1998). Nonspecific binding was measured using 10 µM nisoxetine. It was not possible to test SoRI-6238 concentrations greater than 10^-4 M. Typical total and nonspecific cpms observed for the rat brain transporter binding assays were: SERT (4000; 80), DAT (4000; 90), and NET (1500; 300). Assays using HEK cells expressing hSERT, hNET, or hDAT were conducted as described above, except that no blockers were used, [¹²⁵I]RTI-55 was used as the radioligand for all three transporters, and nonspecific binding was determined with 1 µM indatraline. For the sake of brevity, in the study, hSERT, hNET, or hDAT binding assays refer to assays conducted with HEK cells. Typical total and nonspecific cpms observed for the HEK cell transporter binding assays were: hSERT (4000; 80), hDAT (2000; 75), and hNET (2000; 200).

Kinetic Experiments. Kinetic experiments were conducted with minor modifications of published methods (Nandi et al., 2004). For SERT 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 (TB-1-099, 25 µM; SoRI-6238, 10 µM) was added. This design insured that any effect of the test drug could not be due to interactions with the SERT binding site, since this site would be completely occupied by RTI-55. Triplicate samples were filtered at various time points after the addition of TB-1-099: 10, 30, 50, 80, and 110 min. For the purpose of data analysis, the 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.

In other kinetic experiments we followed methods described by Contreras et al. (1986). Briefly, we measured the association [¹²⁵I]RTI-55 (0.01 nM) to SERT in the absence and presence of competing drugs. Triplicate samples were filtered at eight time points, from 5 min to 2 h. [¹²⁵I]RTI-55 binding surfaces were generated at the same time to permit calculation of the K_d and B_max of SERT in the same preparation of membranes used for the time course. The time course data were fit to the equations described by Contreras for the best-fit estimates of the association (k_on) and dissociation rates (k_off) (Contreras et al., 1986).

Neurotransmitter Uptake Assays. Neurotransmitter uptake inhibition assays were conducted as described elsewhere (Rothman et al., 2001). Freshly removed caudate ([³H]DA uptake) or whole brain minus caudate ([³H]5-HT uptake) were homogenized in 10% ice-cold sucrose with 12 strokes of a handheld 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 1 µM GBR12909 ([³H]DA uptake) or 10 µM fluoxetine ([³H]5-HT). The reactions were stopped after 15-min (for [³H]DA) or 30-min (for [³H]5-HT) incubations by filtering with a Brandel cell harvester over Whatman GF/B filters presoaked in wash buffer (10 mM Tris/HCl, pH 7.4). [³H]NE assays were conducted as described (Rothman et al., 2001). Retained tritium was measured with a Trilux liquid scintillation counter after overnight extraction in 0.6 ml of liquid scintillation cocktail. Typical total and nonspecific cpms observed for the rat brain transporter uptake inhibition assays were: SERT (16,000; 800), DAT (18,000; 1000), and NET (5000; 1300).

Experimental Design. Inhibition curves were generated by displacing a single concentration of [¹²⁵I]RTI-55 (0.01 nM) by 8 to 10 concentrations of drug. For binding surface experiments (Rothman, 1986; Rothman et al., 1991), two different concentrations of radioligand were each displaced by 8 to 10 concentrations of test agents in the absence or presence of various blockers. Similarly, surfaces for [³H]5-HT uptake were generated by displacing two concentrations of [³H]5-HT (2 and 22 nM) each by nine concentrations of 5-HT (2–766 nM) in the absence and presence of TB-1-099. Surfaces for [³H]DA uptake were generated by displacing two concentrations of [³H]DA (10 and 55 nM) by DA (4–1532 nM) in the absence and presence of TB-1-099. The higher concentrations of [¹²⁵I]RTI-55, [³H]DA, or [³H]5-HT were obtained by adding RTI-55, DA, or 5-HT to the radioligand. Dissociation and association experiments were conducted with minor modification of published procedures (Rothman et al., 1991).

Data Analysis and Statistics. Data analysis proceeded as described elsewhere (Rothman et al., 1998). IC₅₀ values were calculated with the MLAB-PC program (Civilized Software, Bethesda, MD) using the two- or three-parameter logistic equation. The two-parameter logistic equation is:

(1)

and the three-parameter logistic equation is:

(2)

where A is the plateau, and N is the slope factor.

For drugs that produced inhibition curves without apparent plateaus, the data were fit to the two parameter logistic equation for the best-fit estimates of the IC₅₀ and N values. For curves with apparent plateaus, the data were fit first to the three-parameter logistic equation to determine the best-fit value of the plateau (A value). Using this value, the inhibition curve was normalized according to the following equation:

(3)

The data were then fit to the two-parameter logistic equation (eq. 1).

Graphs were generated with KaleidaGraph 3.6 software (Abelbeck/Synergy, Reading, PA). Binding surfaces were fit to one- and two-site binding models using MLAB-PC as described elsewhere (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.

Chemicals. [³H]Nisoxetine (SA = 80 Ci/mmol) was obtained from American Radiolabeled Chemicals. [³H]DA (SA = 31.8 Ci/mmol) and [³H]5-HT (SA = 23.7 Ci/mmol) were from PerkinElmer Life and Analytical Sciences (Boston, MA). Thomas Prizansano kindly provided the highly selective DA uptake inhibitor 4-(2-benzhydryloxy-ethyl)-1-(4-nitro-benzyl)piperidine oxalate, synthesized as described (Greiner et al., 2003). SoRI-6238 was synthesized as described (Temple Jr. et al., 1982). The sources of other chemicals are published (Rothman et al., 1998).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Using rat brain membranes, initial studies with TB-1-099 indicated that it weakly inhibited DAT binding (K_i = 440 nM) and was inactive at NET binding, determined with [³H]nisoxetine. As reported in Fig. 1, TB-1-099 partially inhibited SERT binding, with an extrapolated plateau (A value) of 21 ± 4%. In contrast, TB-1-101, which differed from TB-1-099 only in the "R" substituent, completely inhibited SERT binding. SoRI-6238, reported previously (Nandi et al., 2004), also partially inhibited SERT binding, with an extrapolated A value of 34.5%. It was not possible to test higher concentrations of SoRI-6238 because of solubility issues.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Effect of TB-1-099, TB-1-101, and SoRI-6238 on [125I]RTI-55 binding to SERT to rat brain membranes. [125I]RTI-55 (0.01 nM) was displaced by the indicated concentrations of test drug. Each value is the mean of three determinations, which varied by less than 10%. The data for SoRI-6238 are taken from Nandi et al., 2004.

View larger version (17K): [in this window] [in a new window]   Fig. 2. A, effect of indatraline, SoRI-6238, and TB-1-099 on [125I]RTI-55 binding to hSERT expressed in HEK cells. B, effect of TB-1-099 on [125I]RTI-55 binding to hSERT, hDAT, and hNET expressed in HEK cells. [125I]RTI-55 (0.01 nM) was displaced by the indicated concentrations of TB-1-099. Each value is the mean of three determinations that differed by less than 10%. Only the hSERT curve produced a statistically significant plateau (A = 13 ± 2%, F = 55, p < 0.0001). Each point is the mean ± S.E.M. (n = 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Effect of TB-1-099 on [125I]RTI-55 binding to hSERT expressed in HEK cells. A, effect of TB-1-099 on the Kd and Bd of [125I]RTI-55 binding to hSERT. Two concentrations of [125I]RTI-55 (0.01 and 1 nM) were each displaced by 10 concentrations of RTI-55 (0.1 to 35.2 nM) in the absence and presence of various concentrations of TB-1-099 (1, 5, and 25 µM). The data of three experiments were combined and analyzed simultaneously with the control data (144 data points) for the best-fit estimates of the one-site model. The control Bmax = 6910 ± 430 fmol/mg protein and the control Kd = 0.42 ± 0.03 nM. The data for the Kd and Bmax values are reported as a percentage of control. *, p < 0.01 when compared with control (F-test). Each value is ±S.D. B, the effect of TB--099 on the Kd of [125I]RTI-55 binding to hSERT. The data are shown as a percentage of increase compared with the control Kd value of 0.42 nM. The inset shows the effect of TB-1-099 on the apparent Kd assuming that TB-1-099 competitively inhibited hSERT binding with a KI = 1.2 µM.

(4)

The results showed that TB-1-099 decreased the B_max in a dose-dependent manner (Fig. 3A). The effect of TB-1-099 on the apparent K_d was not compatible with competitive inhibition (Fig. 3B). Assuming a competitive K_I of 1.2 µM (the IC₅₀ value for TB-1-099 for hSERT binding reported in Fig. 2), TB-1-099 is predicted to increase the apparent K_d of [¹²⁵I]RTI-55 binding to hSERT in a linear fashion (inset to Fig. 3B). However, the effect of TB-1-099 on the apparent K_d was well described by a sigmoid dose-response curve. Moreover, the magnitude of the TB-1-099-induced increase in the apparent K_d was much lower than predicted according to a competitive inhibition model. In contrast to the effects of TB-1-099, and as expected for a competitive inhibitor, 5 nM indatraline increased the K_d for [¹²⁵I]RTI-55 binding to hSERT from 0.25 ± 0.1 to 1.0 ± 0.1 nM without changing the B_max (data not shown, based on three independent experiments with a total number of 132 data points). The calculated K_i of indatraline was 1.7 nM.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Scatchard plots of the data reported in Fig. 3. As described under Results, the data (264 data points) described in Fig. 3 were fit to the two-site binding model for the best-fit parameter estimates. These parameter estimates were then used to generate predicted Scatchard plots (solid lines) for [125I]RTI-55 binding to hSERT in the presence of 0, 1000, 5000, and 25,000 nM TB-1-099. Scatchard plots of the actual data were generated after the data of the three experiments described in Fig. 3 were averaged and converted to Scatchard format. The best-fit straight lines (dashed) of the actual data for 0 (), 1000 (x), 5000 (+), and 25,000 nM () TB-1-099 are shown above.

The next series of experiments determined the effect of TB-1-099 on the dissociation kinetics of [¹²⁵I]RTI-55 binding to hSERT. Since our previous study showed that SoRI-6238 slowed the dissociation rate of [¹²⁵I]RTI-55 binding to rat brain SERT, we also included SoRI-6238 in these experiments as a positive control. Table 1 reports the experiment design used for these experiments. Briefly, the addition of 1 µM RTI-55 initiated dissociation of [¹²⁵I]RTI-55 hSERT 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 sites, as these were prebound with RTI-55. As reported in Fig. 5A, the dissociation of [¹²⁵I]RTI-55 from hSERT initiated by 1 µM RTI-55 proceeded in a monotonic manner and was well described by a single-component dissociation model (k_off = 0.03 ± 0.002 min^-1). The addition of SoRI-6238 after the addition of RTI-55 slowed the dissociation rate (k_off = 0.01 ± 0.0007 min^-1). The dissociation of [¹²⁵I]RTI-55 initiated by the addition of SoRI-6238 alone was well described by a two-component dissociation model. However, the parameter values had extremely large S.D. values, for example, k_off = 8.6 x 10^-19 ± 0.0006, indicating that the data were in fact too complex or too limited to produce a meaningful fit to a two-component model. Unlike SoRI-6238, TB-1-099 failed to alter RTI-55-induced dissociation of [¹²⁵I]RTI-55 from hSERT (Fig. 5B).

View larger version (28K): [in this window] [in a new window]   Fig. 5. hSERT kinetic dissociation experiments. The experimental design is described in Table 1. Each point is the mean ± S.D. (n = 3). The curves shown are generated by a one-component dissociation model except for the curves for SoRI-6238 alone (A) and TB-1-099 alone (B), which were fit to a two-component dissociation model.

We used the method described by others (Motulsky and Mahan, 1983; Contreras et al., 1986) to compare the effect of SoRI-6238 and indatraline on the association kinetics of [¹²⁵I]RTI-55 binding to hSERT. Indatraline was chosen as an example of a competitive inhibitor. According to this method, a time course of radioligand binding is generated in the absence and presence of competing drug. When the data for radioligand binding in the absence of a competing drug is fit to the appropriate single-ligand equation, it is possible to determine the kinetic constants (k_on and k_off) of the radioligand. The K_d value, determined at the same time by equilibrium binding experiments, should equal the value calculated from the kinetic constants (k_off/k_on). At equilibrium, a competing drug will increase the apparent K_d of the radioligand by a factor equal to (1 + [drug]/K_I). According to Contreras et al. (1986) and Motulsky and Mahan (1983), a competing drug slows the apparent association rate of the radioligand, decreasing the apparent value of k_on. Moreover, the value of k_off must change such that the kinetically calculated apparent K_d (k_off/k_on) increases. In the context of the present experiments, we postulated that indatraline, as a competitive inhibitor of SERT binding, would behave in this way and that TB-1-099, as an allosteric modulator, would not.

View larger version (27K): [in this window] [in a new window]   Fig. 6. hSERT kinetic association experiments. [125I]RTI-55 (0.01 nM) binding to SERT was assessed at various time points in the absence and presence of the indicated test drugs, as illustrated for 1.5 and 15 nM TB-1-099 (A). Similar time courses were run using 2 and 10 nM indatraline (B). Each value is the mean ± S.D. (n = 3). See Table 2 for the data analysis.

As reported in Fig. 7, TB-1-099 inhibited [³H]DA uptake (IC₅₀ = 4190 ± 340 nM) about 10 times more potently than [³H]5-HT uptake (IC₅₀ = 56,600 ± 5540 nM), without any evidence of a plateau. TB-1-099 was inactive at inhibiting [³H]NE uptake (IC₅₀ > 75 µM, data not shown). The K_I values for indatraline, determined at the same time as a positive control, were [³H]DA uptake (7.4 ± 0.5 nM), [³H]5-HT uptake (4.5 ± 0.4 nM), and [³H]NE uptake (11 ± 1 nM). To determine whether TB-1-099 inhibited [³H]neurotransmitter uptake in a competitive manner, uptake surfaces were generated as described in Materials and Methods, allowing us to determine the effect of TB-1-099 on the V_max and K_m of [³H]DA and [³H]5-HT uptake. TB-1-099 competitively inhibited [³H]DA uptake, as indicated by a statistically significant increase in the apparent K_m without any change in the V_max (Table 3). The calculated K_i of TB-1-099 for inhibiting [³H]DA uptake was 405 nM. In contrast, 50 µM TB-1-099 decreased the V_max for [³H]5-HT uptake by about 50%, with a minimal but significant increase in the K_m value. Converting these data to Scatchard format (Fig. 8) clearly showed that TB-1-099 noncompetitively inhibited [³H]5-HT uptake but not [³H]DA uptake. Indatraline, included as a positive control, acted as a competitive inhibitor, with a calculated K_I value of 2.1 nM.

View larger version (19K): [in this window] [in a new window]   Fig. 7. TB-1-099-mediated inhibition of [3H]DA (IC50 = 318 ± 30 nM) and [3H]5-HT (IC50 = 56,600 ± 5540 nM) uptake by rat brain synaptosomes. Uptake assays were conducted as described in Materials and Methods. The individual data points derived from three experiments are plotted above.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Scatchard plots of TB-1-099-mediated inhibition of [3H]DA (B) and [3H]5-HT (A) uptake. "V" refers to the rate of uptake, measured in fmol/mg protein min-1. The data of the uptake binding experiments described in Table 3 were averaged and converted to Scatchard format. The standard deviations of the averaged V values were less than 10% of the mean. For the [3H]5-HT data, the 5-HT concentration of the 14 data points ranged from 3.64 to 108 nM. For the [3H]DA data, the DA concentration of the 20 data points ranged from 14 to 1587 nM.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Based on our experience screening hundreds of compounds in biogenic amine transporter binding assays (Prisinzano et al., 2004), with few exceptions most agents inhibited rat brain SERT binding with slope factors (Hill coefficients) close to 1, a finding strongly suggestive of a binding process governed by simple mass action kinetics. In some cases, we observed that a few compounds had slope factors in the range of 0.80 and were able to resolve two apparent binding SERT binding sites in rat brain cortex using [¹²⁵I]RTI-55 (Silverthorn et al., 1995). SoRI-6238 was the first compound we came across that inhibited [¹²⁵I]RTI-55 binding to rat brain SERT in a qualitatively different manner (Nandi et al., 2004) by virtue of a prominent partial inhibitory profile. Although this pattern might be consistent with the existence of two binding sites, the other lines of evidence that SoRI-6238 allosterically modulated rat brain SERT binding indicated that it would be incorrect to interpret the data in this manner.

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), as well between -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. reported evidence for allosteric modulation of [³H](S)-citalopram binding (Chen et al., 2005).

We initially examined the interaction of TB-1-099, a GBR12909 analog, with rat brain biogenic amine transporters as part of our routine screening efforts (Prisinzano et al., 2004). The fact that TB-1-099 partially inhibited rat brain SERT binding (Fig. 1) provided the first clue that this compound behaved differently than most agents at SERT. Moreover, the observation that changing the R substituent from CF₃ to CH₃"normalized" the SERT inhibition curve suggests that it may be possible, in the future, to determine the structural requirements for the type of partial inhibition observed with TB-1-099. At the present time, we do not have sufficient structure-activity data to hypothesize why such a small change in the structure of TB-1-099 changes its interaction the SERT.

Having gathered initial evidence that TB-1-099 could partially inhibit rat brain SERT binding, we conducted the rest of the study using hSERT expressed in HEK cells, since this model system reduces the probability that the partial inhibition might be due to the existence of two distinct types of SERT binding sites. Although TB-1-099 partially inhibited [¹²⁵I]RTI-55 to hSERT, the plateau was lower than observed in rat brain. This also occurred with SoRI-6238, which did not display a plateau over the concentration examined. The reason for these differences will require additional research. The ability of TB-1-099 to partially inhibit [¹²⁵I]RTI-55 binding to hSERT appeared to be selective for hSERT, since TB-1-099 partially inhibited [¹²⁵I]RTI-55 binding to hSERT but not to hDAT or to hNET (Fig. 2). Interestingly, TB-1-099 was inactive at inhibiting [³H]nisoxetine binding to the rat brain NET and much more potent at [¹²⁵I]RTI-55 binding to hNET. This likely reflects the fact that [³H]nisoxetine underestimates the potency of compounds at NET (Reith et al., 2005).

Saturation binding experiments of [¹²⁵I]RTI-55 binding to hSERT, in the absence and presence of various concentrations of TB-1-099, demonstrated that TB-1-099 noncompetitively inhibited SERT binding (Figs. 3A and 4). Fitting the data to a two-site binding model failed to produce a set of best-fit parameter estimates that described the observed data well, both quantitatively and qualitatively. In particular, the two-site model clearly predicts curvilinear Scatchard plots in the presence of 5000 and 25,000 nM TB-1-099 that extrapolate to the same x-intercept as the control Scatchard plot. This was not observed. Moreover, TB-1-099 increased the apparent K_d but not to the degree expected if the interaction was directly competitive. Indeed, increasing the TB-1-099 concentration from 5 to 25 µM increased the K_d by only 30%, rather than the expected 422%. These observations support the hypothesis that the interaction of TB-1-099 with [¹²⁵I]RTI-55 binding to hSERT is not consistent with competitive inhibition at either one or two binding sites, and supports the hypothesis that TB-1-099 allosterically modulates [¹²⁵I]RTI-55 binding to hSERT.

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. As described in our previous study (Nandi et al., 2004), we could not use the dilution method because a 100-fold dilution produced an initial dissociation followed by a reassociation of [¹²⁵I]RTI-55 binding to hSERT. 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 (TB-1-099 or SoRI-6238) were added 10 min after the RTI-55, insuring that any effect observed could not be due to interactions at the RTI-55 hSERT binding site (see Table 1). Unlike SoRI-6238, which slowed the dissociation of [¹²⁵I]RTI-55 from hSERT, TB-1-099 had no effect. The results reported here for SoRI-6238 extend that of our previous study, which used rat brain membranes.

We have used an experimental approach to association binding experiments pioneered by others (Motulsky and Mahan, 1983; Contreras et al., 1986) to detect the presence of allosteric interactions. This method permits the calculation, from an association time course, of the kinetic constants k_on and k_off. When the time course is conducted in the presence of a competitive inhibitor, the competing drug slows the apparent association rate of the radioligand, decreasing the apparent value of k_on. Moreover, the value of k_off must change such that the kinetically calculated apparent K_d (k_off/k_on) increases. The kinetically calculated apparent K_d can then be compared with the actual apparent K_d determined at the same time. For a competitive inhibitor, the kinetically calculated K_d and the actual K_d should agree fairly well. This is what we observed here for the competitive inhibitor indatraline (Table 2) and in our previous study using citalopram (Nandi et al., 2004). TB-1-099 did not behave like a competitive inhibitor, since increasing its concentration 10-fold increased the kinetically calculated K_d only 2.2-fold, not the expected 6-fold. Although these data support the hypothesis that TB-1-099 does not interact with hSERT binding in a competitive manner, it does not represent direct evidence of an allosteric interaction.

A final piece of evidence that supports an allosteric interaction between TB-1-099 and SERT is that TB-1-099 noncompetitively inhibited [³H]5-HT uptake. As observed in the binding assays (Fig. 2), the apparent allosteric effect appeared to be selective for SERT, since TB-1-099 competitively inhibited [³H]DA uptake. The fact that TB-1-099 noncompetitively inhibits [¹²⁵I]RTI-55 binding to hSERT and [³H]5-HT uptake suggests that TB-1-099, acting via an allosteric site, regulates the availability of SERT binding sites and therefore the functional capacity of the transporter. The relationship between oligomerization of hSERT and the phenomena observed here remains to be determined (Schmid et al., 2001).

As noted in the Introduction, reports over the years hint of an allosteric binding site associated with SERT. Although compelling evidence for an allosteric modulatory site associated with SERT will likely await the development of a high-affinity ligand for the site, the data presented here and in our previous study (Nandi et al., 2004) present strongly suggestive evidence for such an allosteric modulatory site. The implications of such a site for the actions of antidepressants and drugs of abuse, such as cocaine, remains to be determined.

	Acknowledgements

 
HEK cells expressing hNET and hDAT were kindly provided by Dr. Randy Blakely (Vanderbilt University School of Medicine). The hNET cells originated in the laboratory of Dr. Susan Amara (University of Pittsburgh School of Medicine). The hDAT cells originated in the laboratory of Dr. Marc Caron (Duke University Medical Center). The hSERT cells were kindly provided by Dr. Aaron J. Janowsky (Oregon Health and Science University). We acknowledge the expert technical assistance of John H. Partilla, who prepared the [¹²⁵I]RTI-55, and also Dr. F. Ivy Carroll (Research Triangle Institute International), who provided the precursor used in the preparation of [¹²⁵I]RTI-55.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.084376.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; SERT, 5-HT transporter; RTI-55, 3-(4'-iodophenyl)tropan-2-carboxylic acid methyl ester; hNET, cloned human norepinephrine transporter; hDAT, cloned human dopamine transporter; NE, norepinephrine; DA, dopamine; SoRI-6238, ethyl 5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-ylcarbamate; SoRI-9804, 4-[(diphenylmethyl)-amino]-2-phenylquinazoline; GBR12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine; TB-1-099, 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-piperidine; hSERT, cloned human 5-hydroxytryptamine transporter; HEK, human embryonic kidney; BB, binding buffer; SA, specific activity; TB-1-101, 4-{2-[bis-(4-fluoro-phenyl)-methoxy]-ethyl}-1-(2-methyl-benzyl)-piperidine oxalate.

Address correspondence to: Dr. Richard B. Rothman, CPS, DIR, NIDA, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: rrothman{at}intra.nida.nih.gov

	References

Top Abstract Materials and Methods Results Discussion References

 

Akunne HC, de Costa BR, Jacobson AE, Rice KC, and Rothman RB (1992) [³H]Cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: allosteric modulation by paroxetine. Neurochem Res 17: 1275-1283.[CrossRef][Medline]

Amara SG and Kuhar MJ (1993) Neurotransmitter transporters: recent progress. Annu Rev Neurosci 16: 73-93.[Medline]

Andersson A and Marcusson J (1989) Inhibition and dissociation of [3H]paroxetine binding to human platelets. Neuropsychobiology 22: 135-140.[CrossRef][Medline]

Chang AS and Chang SM (1999) Nongenomic steroidal modulation of high-affinity serotonin transport. Biochim Biophys Acta 1417: 157-166.[Medline]

Chen F, Larsen MB, Neubauer HA, Sanchez C, Plenge P, and Wiborg O (2005) Characterization of an allosteric citalopram-binding site at the serotonin transporter. J Neurochem 92: 21-28.[CrossRef][Medline]

Contreras ML, Wolfe BB, and Molinoff PB (1986) Kinetic analysis of the interactions of agonists and antagonists with beta adrenergic receptors. J Pharmacol Exp Ther 239: 136-143.[Abstract/Free Full Text]

Gorman JM and Kent JM (1999) SSRIs and SMRIs: broad spectrum of efficacy beyond major depression. J Clin Psychiatry 60 (Suppl 4): 33-38.[Medline]

Greiner E, Prisinzano T, Johnson IE, Dersch CM, Marcus J, Partilla JS, Rothman RB, Jacobson AE, and Rice KC (2003) Structure-activity relationship studies of highly selective inhibitors of the dopamine transporter: N-benzylpiperidine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine. J Med Chem 46: 1465-1469.[CrossRef][Medline]

Motulsky HJ and Mahan LC (1983) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol 25: 1-9.

Nandi A, Dersch CM, Kulshrestha M, Ananthan S, and Rothman RB (2004) Identification and characterization of a novel allosteric modulator (SoRI-6238) of the serotonin transporter. Synapse 53: 176-183.[CrossRef][Medline]

Prisinzano T, Rice KC, Baumann MH, and Rothman RB (2004) Development of neurochemical normalization ("agonist substitution") therapeutics for stimulant abuse: focus on the dopamine uptake inhibitor, GBR12909. Curr Med Chem 4: 47-59.

Reith ME, Wang LC, and Dutta AK (2005) Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity. J Neurosci Methods 143: 87-94.[CrossRef][Medline]

Rothman RB (1986) Binding surface analysis: an intuitive yet quantitative method for the design and analysis of ligand binding studies. Alcohol Drug Res 6: 309-325.

Rothman RB and Baumann M (2002a) Therapeutic and adverse actions of serotonin transporter substrates. Pharmacol Ther 95: 73-88.[CrossRef][Medline]

Rothman RB and Baumann MH (2002b) Serotonin releasing agents. Neurochemical, therapeutic and adverse effects. Pharmacol Biochem Behav 71: 825-836.[CrossRef][Medline]

Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI, and Partilla JS (2001) Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39: 32-41.[CrossRef][Medline]

Rothman RB, Dersch CM, Carroll FI, and Ananthan S (2002) Studies of the biogenic amine transporters. VIII: identification of a novel partial inhibitor of dopamine uptake and dopamine transporter binding. Synapse 43: 268-274.[CrossRef][Medline]

Rothman RB, Reid AA, Mahboubi A, Kim C-H, de Costa BR, Jacobson AE, and Rice KC (1991) Labeling by [³H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by ligands. Mol Pharmacol 39: 222-232.[Abstract]

Rothman RB, Silverthorn ML, Glowa JR, Matecka D, Rice KC, Carroll FI, Partilla JS, Uhl GR, Vandenbergh DJ, and Dersch CM (1998) Studies of the biogenic amine transporters. VII. Characterization of a novel cocaine binding site identified with [¹²⁵I]RTI-55 in membranes prepared from human, monkey and guinea pig caudate. Synapse 28: 322-338.[CrossRef][Medline]

Rudnick G and Clark J (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters (Review). Biochim Biophys Acta 1144: 249-263.[Medline]

Schmid JA, Just H, and Sitte HH (2001) Impact of oligomerization on the function of the human serotonin transporter. Biochem Soc Trans 29: 732-736.[CrossRef][Medline]

Silverthorn ML, Dersch CM, Baumann MH, Cadet JL, Partilla JS, Rice KC, Carroll FI, Becketts KM, Brockington A, and Rothman RB (1995) Studies of the biogenic amine transporters. V. Demonstration of two binding sites for the cocaine analog [¹²⁵I]RTI-55 associated with the 5-HT transporter in rat brain membranes. J Pharmacol Exp Ther 273: 213-222.[Abstract/Free Full Text]

Sur C, Betz H, and Schloss P (1998) Distinct effects of imipramine on 5-hydroxytryptamine uptake mediated by the recombinant rat serotonin transporter SERT1. J Neurochem 70: 2545-2553.[Medline]

Temple C Jr, Wheeler GP, Elliott RD, Rose JD, Kussner CL, Comber RN, and Montgomery JA (1982) New anticancer agents: synthesis of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines). J Med Chem 25: 1045-1050.[CrossRef][Medline]

Uhl GR and Johnson PS (1994) Neurotransmitter transporters: three important gene families for neuronal function. J Exp Biol 196: 229-236.[Abstract/Free Full Text]

Zohar J and Westenberg HG (2000) Anxiety disorders: a review of tricyclic antidepressants and selective serotonin reuptake inhibitors. Acta Psychiatr Scand Suppl 403: 39-49.[Medline]

This article has been cited by other articles:

				J. J. Pariser, J. S. Partilla, C. M. Dersch, S. Ananthan, and R. B. Rothman Studies of the Biogenic Amine Transporters. 12. Identification of Novel Partial Inhibitors of Amphetamine-Induced Dopamine Release J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 286 - 295. [Abstract] [Full Text] [PDF]

				R. B. Rothman, D. L. Murphy, H. Xu, J. A. Godin, C. M. Dersch, J. S. Partilla, K. Tidgewell, M. Schmidt, and T. E. Prisinzano Salvinorin A: Allosteric Interactions at the {micro}-Opioid Receptor J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 801 - 810. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.084376v1 314/2/906    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nightingale, B. Articles by Rothman, R. B. Search for Related Content PubMed PubMed Citation Articles by Nightingale, B. Articles by Rothman, R. B.