Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3β-aryltropanes with 2β-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2β-Ph2COCH2-3β-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2β compounds increased locomotion, only the 2β-(4-ClPh)PhCOCH2-3β-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2β- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.
Reuptake of dopamine (DA) by the dopamine transporter (DAT) regulates the temporal dynamics of DA synaptic neurotransmission (Rice and Cragg, 2008). Standard DAT inhibitors block DA uptake and in laboratory animals stimulate locomotor activity, fully substitute for cocaine in subjects trained to discriminate cocaine from saline injections, and are self-administered (reviewed by Johanson and Fischman, 1989; Tanda et al., 2009; Schmitt et al., 2013). The relationship between DAT binding affinities and potencies for behavioral effects among DAT inhibitors is the basis for the hypothesis that inhibition of DA uptake mediated by actions at the DAT is the basis for the abuse liability of cocaine, and that compounds with affinity for the DAT will have behavioral effects like those of cocaine (Kuhar et al., 1991).
Contrary to the DAT hypothesis, several groups of compounds with DAT affinity have in vivo or in vitro effects that are distinct from those of standard DAT inhibitors such as cocaine (reviewed by Reith et al., 2015). These “atypical” DAT inhibitors include benztropine (BZT) and its analogs, which consist of the tropane ring of cocaine and the diphenylether moiety common to the piperazine class of DA uptake inhibitors such as GBR 12909 (Van der Zee et al., 1980). BZT analogs selectively bind to the DAT over the other monoamine transporters and inhibit DA uptake in vitro, yet produce behavioral effects distinct from those of cocaine (Katz et al., 1999). BZT analogs are not self-administered in laboratory animals and selectively decrease cocaine self-administration when administered before experimental sessions (Hiranita et al., 2009; Woolverton et al., 2000; Woolverton et al., 2001).
Various mechanisms have been suggested for atypical actions of DAT inhibitors, including affinity for off-target sites such as the muscarinic M1 receptors, which could interfere with the expression of a cocaine-like behavioral profile. However, several studies have suggested that antagonist actions at M1 sites contribute minimally if at all to the atypical effects of BZT analogs (see review by Reith et al., 2015). More recently, it has been suggested that compounds with sigma receptor (σR) antagonist effects along with their affinity for the DAT will exhibit atypical DAT inhibitory effects similar to BZT (Hiranita et al., 2011). A number of previous studies indicated that σR antagonists can block several effects of cocaine, including locomotor stimulation, sensitization, and place conditioning (see review by Katz et al., 2011), and it is noteworthy that BZT analogs also have affinity for σRs (Katz et al., 2004; Li et al., 2011).
Another potential mechanism for atypical actions of DAT inhibitors is the stabilization of cytosol-facing conformations resulting from ligand binding (see review by Reith et al., 2015). For example, Loland et al. (2008) proposed a correlation between conformational states stabilized with DAT binding and the behavioral effects of a diverse group of DAT ligands. Cocaine binds preferentially to an “outward-facing” conformation, in which the primary ligand binding pocket is accessible only from the DAT extracellular face. In contrast, BZT and several analogs can bind and stabilize an “inward-facing” conformation, in which the transporter is open to the cytoplasm. It was shown that compounds stabilizing the inward-facing DAT conformation, like BZT, did not fully substitute for cocaine in rats trained to discriminate cocaine from saline injections, and did not stimulate locomotor activity to the same degree as cocaine (Loland et al., 2008). However, a more recent study showed that RTI-371, a 2-isoxazol-3-phenyltropane cocaine derivative, has behavioral effects similar to the BZT analogs, but stabilizes an outward-facing DAT conformation like cocaine (Hiranita et al., 2014).
Madras et al. (2006) reported that substitutions at the C-2 position of the BZT tropane ring (Fig. 1) decrease affinity for the M1 muscarinic receptor. Additionally, analogs of cocaine exhibit a high degree of tolerance for C-2 substitutions of the tropane ring, as substituents with considerable steric bulk typically do not interfere with cocaine-like pharmacological activity (Carroll et al., 1992; Xu et al., 2004). As the diphenylether moiety of the BZT analogs was shown to be critical for their deviation from typical cocaine-like activity, aryltropane analogs with diphenylether substituents at the C-2 position were of interest. Xu et al. (2004) predicted and confirmed that BZT analogs with C-2 substitutions have less affinity for M1 receptors than the unsubstituted compounds, but retain activity at DAT. In the present study, we report atypical behavioral effects of a series of 2-substituted 3β-aryltropane cocaine analogs (Xu et al., 2004) and explored potential mechanism for their atypical effects.
Materials and Methods
DAT Binding Assay.
Tissue was dissected and homogenized in buffer using a Brinkman Polytron (at setting 6 for 20 seconds) and subsequently centrifuged at 20,000g for 10 minutes at 4°C. The resulting pellet was resuspended in buffer, recentrifuged, and suspended in buffer again to a concentration of 10 mg/ml (original wet weight, OWW). Incubations were conducted in assay tubes containing 0.50 ml of buffer, 0.50 nM radioligand, tissue, and various concentrations of inhibitors. See Table 1 for details.
σ1R and σ2R Binding Assay.
Guinea pig brain was used because of the relatively higher density of σRs in that tissue compared with rat (Nguyen et al., 1996). Tissue was thawed on ice, homogenized (with a glass and Teflon apparatus) in buffer, and subsequently centrifuged at 800g for 10 minutes at 4°C. The supernatant was collected into a clean centrifuge tube and the remaining pellet was resuspended by vortex in 10 ml of buffer and centrifuged at 800g for 10 minutes at 4°C. The supernatants were pooled and centrifuged at 50,000g for 15 minutes at 4°C. The remaining pellet was resuspended 80 mg/ml, OWW, in buffer and vortexed. The tissue suspension was incubated at 25°C for 15 minutes, and then centrifuged at 50,000g for 15 minutes. The supernatant was decanted and the pellet was gently resuspended in buffer to 80 mg/ml, OWW. Incubations were conducted in polypropylene assay tubes containing 0.50 ml of buffer, 1.4 nM radioligand (and 200 nM (+)-pentazocine for σ2 binding), tissue, and various concentrations of inhibitors. See Table 1 for details.
The reactions in all binding assays were started with the addition of tissue and terminated by rapid filtration through Whatman GF/B filters (presoaked in 0.050% polyethylenimine) using a Brandel Cell Harvester (Brandel Instruments, Gaithersburg, MD). The filters were washed twice with 5.0 ml of cold buffer and transferred to scintillation vials, to which Beckman Ready Safe scintillation cocktail (3.0 ml; PerkinElmer, Waltham, MA) was added. The vials were assessed for radioactivity the next day using a Beckman LS6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton, CA) at 50% efficiency. Assays were typically conducted as three or more independent experiments, each performed with triplicate tubes.
The IC50 values for the displacement of radioligands were computed using a nonlinear, least-squares regression analysis for competitive binding (GraphPad Prism, San Diego, CA). Inhibition constants (Ki values) were calculated using the Cheng-Prusoff equation (Cheng and Prusoff, 1973), with IC50 of inhibitors used in the assay and the Kd value of the radioligand previously determined in this laboratory.
An enantiomeric pair of compounds (LX-19 and LX-20), one with and one without cocaine-like locomotor-stimulant effects, was screened for activity at various receptor sites by examining their competition with the appropriate radioligands (ProfilingScreen procured from MDS Pharma Services, Eurofin Panlabs Washington, Bothell, Washington). The screen consisted of assays designed to assess the activity of the compounds at 35 mammalian receptors. Each compound was tested in each assay at a 10 μM concentration. Vehicle and reference standards were assessed concurrently with each assay. Details of the procedures and targeted sites are provided in Table 2.
Estimating affinity of the compounds from radioligand displacement data at single concentrations follows a practice used by Kosterlitz et al. (1973) to estimate potencies from single concentrations of opioids inhibiting electrically driven contractions of guinea pig ileum. For sites at which the compounds produced displacement between 10 and 90%, an IC50 value was derived from linear interpolation, assuming a typical displacement curve spanning a 100-fold domain of concentrations. For sites at which the displacement was >90%, IC50 values were estimated to be <1 μM. The compound was considered inactive at sites for which the displacement by 10 μM was <10%. The Cheng-Prusoff equation (Cheng and Prusoff, 1973) was subsequently used to derive Ki values of compounds at a particular site from the estimated IC50 values, using Kd values of radioligands provided by the vendor.
Male Swiss-Webster mice (Taconic Farms, Germantown, NY) for locomotor activity assessments and Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) in cocaine discrimination studies were acclimated to the animal colony for at least 1 week before use. During this time, food and tap water were freely available. Mice were housed in groups of three to four per cage and rats were housed singly and maintained at approximately 320 g body weight by adjusting their daily food ration (∼15 g/d). The humidity- and temperature-controlled colony rooms were maintained on a 12:12-hour light/dark cycle with lights on at 0700 hours. The animals were cared for in accordance with the guidelines of the National Institutes of Health Animal Care and Use Program and the National Institute on Drug Abuse Intramural Research Program Animal Program, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Subjects were placed individually in clear acrylic chambers (40 cm3) for the measurement of horizontal (locomotor/ambulatory) activity. The acrylic chambers were contained within monitors (Omnitech Electronics, Columbus, OH) equipped with infrared light sources mounted 2.5 cm apart along two perpendicular sides. Light-sensitive detectors were mounted on the opposite wall, in line with the light sources. One locomotor activity count was registered with each interruption of light to the detectors. Injections were administered intraperitoneally immediately before the subjects were placed in the apparatus for 1 hour, with each drug dose studied in eight subjects. Locomotor activity counts were totaled separately in the first and second 30 minutes. Whether locomotor activity was stimulated above that obtained with vehicle was determined in each 30-minute portion of the session by analysis of variance (ANOVA) and post-hoc comparisons. Subjects were each used only once.
Details of the apparatus and procedures were as described previously (Katz et al., 1999). Briefly, subjects were trained to press response levers under a 20-response fixed ratio (FR 20) schedule of food reinforcement. When saline was injected immediately before sessions, responses on only one lever were reinforced; with cocaine injections, responses on the other lever only were reinforced. The assignment of saline and cocaine to right or left levers was counterbalanced across subjects. The session started with the illumination of the house light and the light-emitting diodes 5 minutes after subjects were placed in the chamber. Only responses on the appropriate lever were reinforced and responses on the inappropriate lever reset the FR response requirement. Food presentations were followed by 20-second time out periods during which all lights were off and responding had no scheduled consequences. Sessions ended after 20 food presentations or 15 minutes after their start, whichever occurred first. Training sessions with saline (S) and cocaine (C) injections were conducted in a double-alternation sequence [e.g., ...SCCS...].
Testing was initiated when performances met or exceeded training criteria of at least 85% appropriate responding overall, and during the first FR 20 of the session over four consecutive sessions. Tests were conducted with different doses of cocaine, doses of the aryltropane analogs, or combinations of doses, after which training resumed. Subsequent testing was conducted only if the subject met the above criteria on both of the immediately preceding training sessions. Test sessions were identical to training sessions with the exception that 20 consecutive responses on either lever were reinforced.
Overall response rate and the percentage of responses occurring on the cocaine-appropriate lever were calculated for each subject and averaged. If less than half of the subjects responded at a particular dose, the mean value was not determined for percentage of cocaine appropriate responding. At least 80% cocaine- or saline-appropriate responding was considered similar to the training dose of cocaine or saline, respectively. Levels of cocaine appropriate responding between 20 and 80% were considered partial substitution.
(-)-Cocaine HCl was purchased from MilliporeSigma (St. Louis, MO), and analogs of phenyltropane were synthesized at the University of New Orleans (Xu et al., 2004). Figure 1 shows reference compounds and structures of 2-substituted 3β-aryltropane compounds used in this study. The substitutions examined in the present study were either on the diphenylether moiety or para substitutions on the 3-phenyl moiety. All drugs were dissolved in 0.9% NaCl, and administered intraperitoneally at 1 ml/kg (rat) or 1 ml/100 g (mouse) body weight in doses as indicated.
Ligand-Induced DAT Cysteine Accessibility.
Human embryonic kidney 293 (HEK293) cells stably transfected with T316C/C306A human DAT were seeded into polyethylenimine-coated six-well plates and cultured to confluency. Cells were washed with cold phosphate-buffered saline (PBS) with 0.10 mM CaCl2 and 1.0 mM MgCl2, pH 7.1 (PBSCM), and incubated with DAT inhibitors in PBSCM for 20 minutes at 4°C. Cells were then further incubated with 1.0 mg/ml maleimide-PEO2-biotin (Pierce Biotechnology, Rockford, IL) in the presence of DAT inhibitors for 30 minutes at 4°C, followed by quenching with 100 mM cysteine in PBSCM for 15 minutes at 4°C. Cells were then washed, harvested, and lysed in TNE lysis buffer (Tris 10 mM, NaCl 150 mM, EDTA 1.0 mM, 1.0% Triton X-100, protease inhibitors, pH 7.5) for 1 hour at 4°C, followed by a 20-minute centrifugation of 18,000g. The lysates were incubated with 60 μl of Pierce NeutrAvidin Agarose beads (ThermoFisher Scientific, Grand Island, NY) overnight at 4°C. After beads were washed three times with TNE buffer, biotinylated proteins were eluted with sodium dodecyl sulfate sample buffer, separated by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with rabbit antisera for DAT (Hong and Amara, 2010). Mean densities of chemiluminescent DAT bands were quantified using the NIH ImageJ software and normalized to percent of vehicle.
Molecular Dynamics Simulation of DAT-Ligand Interactions.
Starting coordinates for the inward-open and outward-open rat DAT (rDAT) homology models were obtained from a previously developed homology model on the basis of the 2A65 LeuT crystal structure (Yamashita et al., 2005). The rDAT model was subjected to 600 nanoseconds (ns) of all-atom molecular dynamics (MD). The root mean squared deviation (RMSD) was measured for backbone atoms of TMs 1–12 with reference to the outward-open crystal structure of dDAT (4M48) (Penmatsa et al., 2013) and the inward-open crystal structure of LeuT (3TT3) (Krishnamurthy and Gouaux, 2012). Cocaine was docked into the outward-open rDAT homology model and BZT was docked into the inward-open rDAT homology model. One nanosecond of MD was performed on cocaine bound to DAT, after which cocaine was removed from the binding pocket and LX-10 and LX-11 were independently docked into the outward-open DAT structure. Interactions between BZT, cocaine, LX-10, and LX-11 were evaluated within their respective DAT models after 35 ns of all-atom MD.
The isobaric-isothermal (NPT) ensemble in the Nanoscale Molecular Dynamics (NAMD) program (v2.10b1) was used to perform all MD calculations using a time step of 2 femtoseconds, and periodic boundary conditions. Force field parameters for the protein, lipids, and water were defined using the CHARMM36 force field. Ab initio–derived force field parameters were previously developed and used for assigning parameters for cocaine. CHARMM General Force Field (CGenFF) parameters for BZT and the 2-substituted aryltropanes were obtained from the ParamChem webserver (https://www.paramchem.org/). Langevin dynamics and the damping Langevin piston were used to maintain 310 K temperature and 1 atm pressure, respectively. A 20-picosecond (ps) energy minimization was performed to eliminate steric clashes, followed by 1 ns of MD with harmonic restraints placed on the protein backbone atoms. The atoms’ restraining potential was scaled from 10 to 0 kcal/mol per Angstrom. All-atom MD was performed with restraints removed from the backbone atoms for 35 ns. The RMSD of each DAT-inhibitor complex was evaluated after the 35-ns MD with reference to the initial DAT coordinates.
Radioligand Binding Assays.
The Ki values determined with the 3β-aryltropanes with 2β-substitutions at the DAT ranged from 18.9 for LX-10 to 68.0 nM for LX-19 (Table 3). These values represent affinities as much as 3-fold higher than those reported previously (Xu et al., 2004) owing to the use of a sucrose-phosphate rather than a HEPES buffer (unpublished observations). Additionally, the 2β-substituted analogs had affinities that were from 10- to 25-fold greater than the respective analogs with α-substitutions. In contrast to the β-substituted analogs, the α-substituted analogs were generally not selective for DAT over serotonin transporter (SERT), on the basis of previously published affinities for the SERT (Xu et al., 2004). None of the compounds had better than micromolar affinity for either σ1R or σ2R binding sites, and those affinities were not appreciably different among the enantiomers (Table 3).
A pair of enantiomers (LX-19 and LX-20) were screened for affinity at 35 mammalian binding sites at a 10 μM concentration of either compound. Compounds were considered inactive when they produced less than 10% displacement at all of the screened sites with the exception of those listed in Table 4. Affinities of LX-19 for those sites were uniformly less than that for the DAT, though reasonable affinity measurements were impossible at adrenergic α2A, 5-HT2A, or sodium channel, site 2, because there was greater than 90% displacement of radiolabel by LX-19 at 10 μM (Table 4). The estimated affinity of LX-20 for histamine H2 receptors was greater than that for the DAT. Additionally, at nine other sites the affinity of LX-20 was comparable to or possibly greater than that for the DAT. Finally, the affinity of the compound at dopamine D2 receptors was less than that at the DAT (Table 4).
Cocaine dose dependently increased ambulatory activity (Fig. 2, squares, repeated in all panels for comparison with the other compounds). The maximum effect of cocaine was approximately 650 counts per min during the first 30 minutes after injection, and was obtained at 59 μmol/kg. All of the 2β-substituted compounds dose-dependently stimulated locomotor activity (Fig. 2, filled circles). The dichloro-substituted compound LX-19 (Fig. 2D) was the most effective and stimulated activity to a maximum similar to that produced by cocaine (t14 = 0.310, P = 0.761). The maximal effects of the other 2β-substituted compounds were statistically less than the cocaine maximum (all t-values ≥ 2.47, P values ≤ 0.027).
In contrast to the compounds with 2β-substituents, those with 2α-substituents generally failed to stimulate locomotor activity (Fig. 2, open symbols). The exception to this was the methyl-substituted LX-22 (Fig. 2E), which increased activity by 163 counts per min. That value was about half and statistically less than (t14 = 3.14, P = 0.007) the maximum produced by its 2β-enantiomer, LX-21. The characteristic failure of the 2α-substituted compounds to stimulate locomotor activity occurred despite the assessment across the range of behaviorally active doses, from those that had no effect to those that substantially decreased activity. This range typically encompassed doses that were at least 10-fold higher than the minimally effective stimulant dose of the corresponding 2β enantiomers.
Cocaine produced a dose-related increase in the percentage of cocaine-appropriate responses (Fig. 3, all top panels, squares). Among the 2β-substituted aryltropanes, LX-10 (Fig. 3A) was the single compound that fully substituted for cocaine, with 92% of responses on the cocaine-appropriate lever at a dose of 12 μmol/kg (Fig. 3A, top). Several of the other 2β-substituted analogs (LX-16, LX-19, LX-21; Fig. 3) produced a degree of cocaine-appropriate responding greater than that found following vehicle injections, though none exceeded 60%. In contrast, the remaining 2β-substituted analogs (LX-13, LX-23) produced maximal substitution for cocaine well below 20% (Fig. 3).
Likewise, none of the 2α-substituted aryltropane analogs produced a level of cocaine-appropriate responding exceeding 20% (Fig. 3). The absence of full substitution for cocaine was obtained despite the assessment across the entire range of behaviorally active doses, from those having no effects to those that virtually eliminated responding (Fig. 3, bottom panels).
Because it is possible that the time frame for the assessment of discriminative-stimulus effects may have failed to capture the maximal effects of the 2-substituted aryltropanes, the effects of two of the β-enantiomers, LX-21 and LX-23, were also examined at 30 and 60 minutes after injection. Increasing the time between treatment and testing did not significantly increase the effectiveness of either compound in substituting for cocaine (Fig. 4, top panels). In addition, at 30 and 60 minutes after injection, the decreases in response rates produced by the drugs were substantially diminished (Fig. 4, bottom panels). The diminished effects on response rates are indicative of diminished pharmacological effects with time, and that further increases in time between injection and the assessment would not yield greater substitution for cocaine.
The compounds LX-13, LX-21, and LX-23 were selected for studies in combination with cocaine owing to their high DAT affinity and their own minimal substitution for cocaine shown in Fig. 3. Each of the 2-substituted aryltropane analogs shifted the dose-effect curve to the left for discriminative-stimulus effects of cocaine, and in a dose-related manner for LX-13 and LX-23 (Fig. 5). The lowest doses studied produced a leftward shift of about 1.5-fold. The higher doses produced shifts of 2.23- and 3.31-fold with LX-13 and LX-23, respectively, which were statistically significant (95% confidence limits of the relative potency values excluded 1.0).
To compare molecular actions of these compounds with those of cocaine at DAT, the conformation of DAT was probed biochemically using the T316C/C306A construct (substitution of threonine 316 to cysteine and cysteine 306 to alanine). This functional DAT construct has a sole cysteine residue (T316C) accessible from the extracellular side, as measured from alkylation of its sulfhydryl side chain by the cell-impermeant maleimide-PEO2-biotin. The reactivity at this position has been shown be altered by inhibitor binding, and is used to indicate a DAT conformation open to the extracellular space (Hong and Amara, 2010). Incubation with saturating concentrations (>100-fold Ki values) of the six 2β-enantiomers significantly increased DAT labeling by maleimide-PEO2-biotin, suggesting a cocaine-like stabilization effect on the outward-open DAT conformation. Additionally, the majority of the 2α-enantiomers (with the exception that LX-15 and LX-20 were inactive) also increased T316C DAT cysteine accessibility, indicating a cocaine-like effect on the DAT conformation (Fig. 6).
Molecular Dynamics Simulation of DAT-Ligand Interaction.
Initial docking of cocaine, LX-10, or LX-11, to the outward-open DAT conformation, and BZT to the inward-open DAT conformation yielded poses in which each inhibitor was able to occupy subsites A, B, C within the binding site (Wang et al., 2013). Interaction between the positively-charged tropane nitrogen atom of each ligand and the TM1 residue D79 was also observed in each pose. Each inhibitor-DAT complex was then embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipid bilayer and solvated using a TIP3P water model with 0.15 M NaCl and 0.15 M KCl.
After 35 ns of MD, the tropane ring of cocaine was still interacting with TM1 residue D79 and TM3 residue Y156 (Fig. 7A). However, the phenyl ring of cocaine was no longer interacting with F325 in a “face-to-edge” interaction. In contrast to cocaine, BZT bound to the inward-open DAT conformation was repositioned by the MD iteration (Fig. 7A). BZT was reoriented toward the intracellular end of the binding pocket, although its tropane ring nitrogen maintained an interaction with D79. Both rings of the diphenyl ether moiety were observed to interact face-to-edge with F76 and F325, allowing the ligand to be stabilized by several hydrophobic interactions within the S1 site.
LX-10 and LX-11 were each, like cocaine, still interacting with TM1 residue D79 and TM3 residue Y156 after 35 ns of MD (Fig. 7A). LX-10 was found to occupy the S1 pocket, with its functional groups overlapping with the placement of cocaine’s functional groups (Fig. 7B). The LX-10 tropane and chlorophenyl ether rings were seen to interact with DAT side-chain residues in a way similar to cocaine. In contrast, the diphenyl ether moiety of LX-10 was seen to interact with F325, as it extends further than the 2-methyl ester group of cocaine. Unlike LX-10 and cocaine, the chlorophenyl ring and one of the diphenyl ether rings of LX-11 displayed a BZT-like DAT interaction pattern. Its second diphenyl ether ring was found to form a π-π interaction with F325, and a new π-π interaction was observed between the chlorophenyl ring of LX-11 and F76, similar to BZT (Fig. 7B). Overall, the positioning of LX-10 in the DAT S1 pocket showed greater similarity to cocaine than did LX-11.
To examine accessibility of the DAT binding pocket from the extracellular side, the distance between two critical residues, R85 on TM1a and D475 on TM10 (Fig. 7C) was measured in the MD simulation. When BZT binds to the inward-open DAT, the average distance was 3.0 ± 0.3 Å, substantially smaller than those for DAT binding to cocaine, LX-10, or LX-11 (6.7 ± 0.6 Å, 5.1 ± 1.4Å, or 5.6 ± 1.0Å, respectively), suggesting BZT is positioned more deeply in the DAT binding pocket (Fig. 7D).
Previous studies demonstrated that the behavioral potencies of DA uptake inhibitors including cocaine are directly related to their DAT binding affinities (Ritz et al., 1987; Bergman et al., 1989). The correlation among those effects is greater than that for behavioral effects and affinities at the other monoamine transporters (Ritz et al., 1987), suggesting that behavioral effects of cocaine-like drugs are attributable to actions at the DAT (Kuhar et al., 1991). However, an increasing number of DAT inhibitors are being found with effects different from those of cocaine (Reith et al., 2015).
The 2-substituted aryltropane analogs examined in the present study had affinity for the DAT; however, virtually all of the compounds had behavioral effects that were substantially different from those of cocaine. Most of the compounds failed to fully substitute in rats trained to discriminate cocaine from saline injections with the 3-chlorophenyl analog (LX-10) as the only exception. The 2α-substituted compounds failed to stimulate locomotor activity, whereas the 2β-substituted compounds did so, but with only one compound (LX-19) as effective as cocaine. Thus, although these 2-substituted aryltropanes bind to the DAT, they produce behavioral effects atypical for drugs with DAT affinity.
The absence of cocaine-like behavioral effects of the 2α-substituted aryltropanes in the present study possibly resulted from their lower DAT affinity compared with the β-enantiomers. However, locomotor effects of the α-enantiomers (with the possible exception of LX-15) were assessed at doses sufficient to accommodate their lower affinity. In the drug-discrimination procedure, the dose domain studied was from inactive doses to those that virtually eliminated responding. Because the pairs of enantiomers were typically equipotent in decreasing response rates it is possible that doses of the α-enantiomers sufficient for cocaine-like effects were greater than those that eliminated responding. The similar potencies for effects on response rates of the enantiomeric pairs and their differences in DAT binding affinities suggest that effects on response rates were not DAT-mediated.
The major structural difference between the present aryltropanes and cocaine-like DAT inhibitors (e.g., Carroll et al., 1992) resides in the diphenyl-ether substitution on the tropane 2-position, which is a significant substituent of atypical DA uptake inhibitors that are structurally related to BZT (van der Zee et al., 1980; Newman et al., 1995) and the phenylpiperazines (e.g., GBR 12909; van der Zee et al., 1980; Andersen, 1989). Many of the members of those structural groups have various effects atypical of standard DAT inhibitors such as cocaine and methylphenidate. Reith et al. (2015) discussed several mechanisms that may contribute to atypical effects of DAT inhibitors, including off-target actions, slow DAT association, and shifts in DAT conformational equilibrium that decrease its opening to the extracellular space.
The pharmacology of the present series of aryltropanes has some similarities to and differences from that of BZT analogs. Many of the BZT analogs have muscarinic M1 antagonist effects (Tanda et al., 2009), an off-target action thought by some to interfere with cocaine-like effects (Tanda et al., 2009; Reith et al., 2015). The present compounds have appreciably lower M1 affinity, and results here are consistent with previous results showing that atypical DAT inhibitors can have effects different from cocaine without having affinity for M1 receptors (Tanda et al., 2009; Reith et al., 2015).
Previous studies indicate that concurrent DAT and σ1R inhibition can alter the effects of DAT inhibitors (e.g., Hiranita et al., 2011). However, the relatively low σ1R binding affinities of the present compounds suggest that actions at that site do not contribute to their atypical effects. The present DAT affinities were obtained in a sucrose-phosphate buffer, which yielded higher affinities than those reported previously for binding in a HEPES buffer, and consequently the selectivity of these compounds for the DAT compared with SERT and norepinephrine transporter (NET) would be greater than that reported previously (Xu et al., 2004). Thus the relatively lower SERT and NET affinities of these aryltropanes are also unlikely to produce effects that interfere with cocaine-like effects. Further, numerous cocaine analogs that share behavioral effects with cocaine bind to SERT and NET, often with affinities as great as those for the DAT (Carroll et al., 1992).
Sites at which the screened compounds were most potent include DA D3, histamine H2, muscarinic M2, and 5-HT1A receptors. Considering these off-target actions with previous findings suggests that none of these is the sole contributor, if there is one at all, to the atypical effects of these compounds. For example, the D3R affinity of LX-20, which did not stimulate locomotor activity, was greater than that for LX-19, which did stimulate activity, and greater than its affinity for the DAT, suggesting a role for D3Rs in the atypical effects of LX-20. However, Li et al. (2011) examined a group of BZT analogs that were considered atypical DAT ligands. Within that group, the compound that was most like cocaine, JHW013, had the greatest affinity for D3Rs. Together, the results suggest that DA D3Rs do not contribute to atypical DAT inhibitor effects.
A previous study indicated that affinity for histamine receptors does not contribute to the atypical effects of BZT analogs (Campbell et al., 2005). Likewise, muscarinic M2 sites seem improbable, as the BZT-derived atypical DAT inhibitors have a wide variety of reported activity at this site. Notably, the N-substituted BZT analog GA-299 had little cocaine-like activity and lacked affinity for the M2 muscarinic site (Li et al., 2011). Previous studies have also found inconsistencies in affinities at 5-HT1 binding sites among N-substituted BZT analogs (Katz et al., 2004; Li et al., 2011).
Several studies (Stathis et al., 1995; Desai et al., 2005a,b; Tanda et al., 2009) suggest that a slow rate of DAT occupancy by various DAT inhibitors can decrease cocaine-like behavioral effects. Slow brain penetration also reduces the reinforcing effectiveness of DA uptake inhibitors (Kimmel et al., 2007; Czoty et al., 2010). It is therefore plausible that slow DAT occupancy rates with the present aryltropanes renders them ineffective in substituting for cocaine in drug-discrimination paradigms early after injection. To assess this, the time courses of two of the β-substituted compounds (LX-21, LX-23) that had minimal cocaine-like effects were assessed. Maximal effects were not enhanced at these later times, suggesting that a slow association at the DAT is not responsible for the present atypical effects.
Because of the limited effectiveness of the present aryltropane analogs compared with cocaine, it was of interest to assess whether these compounds might reduce/antagonize the effects of cocaine. It has been previously reported that the discriminative effects of cocaine can be potentiated by DA uptake inhibitors, both “standard” inhibitors that did (Holtzman, 2001; Li et al., 2006) and those that did not (Tolliver et al., 1999; Katz et al., 2004) fully substitute for cocaine. The present compounds enhanced rather than antagonized the discriminative stimulus of cocaine. Interestingly, compounds that shifted the discriminative effects of cocaine leftward in a previous study (Katz et al., 2004) decreased the self-administration of cocaine (Hiranita et al., 2009), suggesting differences in interactions with cocaine self-administration and discrimination.
Molecular studies have shown that cocaine binding alters DAT residue accessibility to alkylation (Ferrer and Javitch, 1998), and that BZT binding results in a different pattern of DAT residue accessibility (Reith et al., 2001). Recent reports propose that cocaine binding stabilizes an outward-facing DAT conformation (Loland et al., 2008; Hong and Amara, 2010). Further, it was suggested that behavioral effects produced by cocaine-like or atypical DAT inhibitors are related to different conformational states of DAT preferentially induced by these ligands (Loland et al., 2008). We thus examined whether binding of these 2-substituted aryltropane analogs prefers certain conformational states of DAT. Results from the T316C/C306A DAT construct are consistent with previous reports (Hiranita et al., 2014; Reith et al., 2015). The cysteine accessibility of T316C was increased by cocaine, but decreased by BZT. Although the present aryltropane analogs did not induce significant locomotor activity or substitute for cocaine in drug discrimination paradigms, all 2β enantiomers and the 2α enantiomers (with the exception of LX-15 and LX-20) significantly increased DAT cysteine accessibility, as did cocaine, suggesting that their binding stabilized the outward-facing DAT conformation in a manner similar to cocaine.
Molecular dynamics simulations of DAT-ligand interactions suggest that cocaine binds to DAT in the S1 pocket, preferring an outward-open conformation, whereas BZT appears to have been significantly repositioned, occupying a new novel subsite in the DAT S1 pocket, at the same time maintaining several favorable interactions with S1 residues. Combined with the cysteine accessibility data, the results support the notion that BZT binds to DAT in a manner distinct from that of cocaine. Further, both the cysteine accessibility results and the molecular dynamics simulations suggest that LX10 and LX 11, like cocaine, prefer an outward-open DAT conformation. Consideration of these data with the behavioral data suggests that the behavioral effects of DAT inhibitors cannot be fully explained by conformational status assumed by the DAT with ligand binding. As with previous reports (Li et al., 2011; Hiranita et al., 2014), the present results suggest that multiple mechanisms are probably involved in the behavioral effects of atypical DAT inhibitors (Reith et al., 2015).
The authors thank Bettye Campbell and Dawn French for technical support, Lekyla Whitaker for administrative and clerical support, and Dawn French for expert data analysis. We also thank Drs. Brandon Harvey and Bruce Hope for sharing their cell culture facility and biochemical instruments. Portions of this manuscript were presented (by WCH, TAK, LX, SAL, MLT, JLK) at Experimental Biology 2015 conference, March 28–April 1, Boston.
Participated in research design: Hong, Kopajtic, Xu, Lomenzo, Jean, Madura, Surratt, Trudell, and Katz.
Conducted experiments: Hong, Kopajtic, Xu, Lomenzo, Jean, Madura, Surratt, Trudell.
Contributed new reagents or analytic tools: Hong, Xu, Lomenzo, Jean, Madura, Surratt, Trudell.
Performed data analysis: Hong, Kopajtic, Xu, Lomenzo, Jean, Madura, Surratt, Trudell, and Katz.
Wrote or contributed to the writing of the manuscript: Hong, Kopajtic, Lomenzo, Jean, Madura, Surratt, Trudell, and Katz.
- Received November 13, 2015.
- Accepted January 7, 2016.
The work reported herein was supported in various parts by funds as follows: Work by W.C.H., T.A.K., and J.L.K. was supported by the Intramural Research Program of the National Institutes of Health National Institute on Drug Abuse [ZIA DA000103-26]. W.C.H. was also supported by startup funds from Butler University. The work of J.D.M. and C.K.S. was supported by a grant from the National Institute on Drug Abuse [DA027806]. The efforts by J.D.M. and B.J. were funded in part by National Institutes of Health National Science Foundation grants [R01DA027806 and XSEDE MCB060069 supercomputers]. The work of M.L.T., L.X., and S.A.L. was supported by a grant from the National Institute on Drug Abuse [DA11528].
- dopamine transporter
- fixed ratio
- GBR 12909
- half maximal inhibitory concentration
- norepinephrine transporter
- original wet weight
- polyethylene oxide
- phosphate-buffered saline
- PBS with CaCl2 and MgCl2
- serotonin transporter
- sigma receptor
- U.S. Government work not protected by U.S. copyright