Identification of a Novel Allosteric Modulator of the Human Dopamine

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter Shaili Aggarwal,† Xiaonan Liu,† Caitlyn Rice,† Paul Menell,† Philip J. Clark,‡ Nicholas Paparoidamis,§ You-cai Xiao,§ Joseph M. Salvino,§ Andreí a C. K. Fontana,† Rodrigo A. España,‡ Sandhya Kortagere,*,∥ and Ole V. Mortensen*,†

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†

Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States ‡ Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, United States § The Wistar Institute, Philadelphia, Pennsylvania 19104, United States ∥ Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, United States S Supporting Information *

ABSTRACT: The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function. KEYWORDS: Allosteric modulation, cocaine use disorder, dopamine transporter, hybrid structure based method, substituted cysteine scanning accessibility method

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INTRODUCTION The dopamine transporter (DAT) is a member of the solute carrier 6 (SLC6) family of transporters and is embedded in the plasma membrane of presynaptic terminals of dopaminergic neurons in the central nervous system.1,2 DAT plays a key role in controlling the signal amplitude and duration of dopaminergic neurotransmission by removing extracellular dopamine (DA) resulting in decreased levels of DA in the extracellular space. Pharmacological modulation of the DAT will as a result affect neuronal dopaminergic activity. Indeed, DAT is the primary site of action for a number of psychostimulants and recreational drugs, including cocaine, amphetamine, and methamphetamine, which all block or reverse the transport of DA, thereby increasing synaptic dopaminergic neurotransmission.3 © XXXX American Chemical Society

DAT is a 12-transmembrane domain (TMD) protein mediating DA uptake through the coupling to the sodium and chloride gradients. The substrate-translocation is believed to follow an “alternating access” mechanism where the transporter sequentially transitions through several different conformations including outward-open, occluded and inwardopen conformations to transport substrate from extracellular side to the intracellular milieu.4 The discoveries of homologous bacterial (Aquifex aeolicus) leucine transporter (LeuT),5 Drosophila melanogaster DAT (dDAT)6,7 and human serotonin transporter (SERT)8 crystal structures have been pivotal in Received: May 2, 2019 Accepted: June 11, 2019 Published: June 11, 2019 A

DOI: 10.1021/acschemneuro.9b00262 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 1. KM822 interactions with hDAT. (A) molecular model of hDAT represented as ribbons bound to KM822 shown as van der Waals surface and colored atom type (carbon, cyan; oxygen, red; nitrogen, blue; and sulfur, yellow). Transmembrane domains and loop regions contributing to the binding of KM822 are colored as follows: TMD1 = blue, TMD3 = light green, TMD10 = yellow, TMD11 = pink; EL4 = magenta and EL5 = orange; EL6 = mauve while the rest of the protein is colored gray. (B) Five-point receptor based pharmacophore formed by residues W84, D385, D476, R544 and Y548 along with distance restraints are shown. (C) Schematic interaction diagram of KM822 in the allosteric site was generated using ligand interactions module of MOE. Legend details the nature of the interactions.

pathway that dictated pharmacological differences between human transporters and transporters from the parasite Schistosoma mansoni.9,10 We used the hybrid structure-based (HSB) method11 to successfully identify an allosteric modulator of human SERT (ATM7) that interacts with this allosteric site.12 Intriguingly, the site we identified through computational and biochemical studies is similar to the site where the second citalopram molecule later was found in the SERT cocrystal structure. Other functional studies have also pointed to this region as a domain that could have allosteric activity.9 For example, in studies on the bacterial DAT homologue LeuT, a site (S2) was proposed to exist in the extracellular vestibular region that lies in the solvent-accessible pathway connecting the extracellular milieu of the transporter with the orthosteric S1 site.13 This site overlaps partially with the site we have identified in this study. Engaging S2 by substrates is thought to allosterically trigger a conformational change in the LeuT transporter from the substrate-bound occluded state to an inward open state, which facilitates the release of the substrate. In the current study, we target a similar region in DAT that we previously targeted in SERT. Since this site is proximal to the orthosteric site and is networked through a series of hydrogen bonds, we hypothesize that conformational changes induced by small molecules at this site will produce allosteric effects. Indeed, we demonstrate that a small molecule, KM822, identified using the HSB screening, interacts at this site and causes significant changes in the structure and function of the DAT protein. We also show that this molecule affects cocaine

enhancing our knowledge on the structural biology of this family of neurotransmitter transporters. The structure of these transporters includes 12 α-helical TMDs connected with flexible intracellular and extracellular loops. The N- and Ctermini lie in the intracellular region. The high-affinity primary orthosteric substrate binding site, S1, lies at the core of the translocation pathway located between TMD1 and TMD6. Interestingly, besides the S1 site where substrates and psychostimulants bind, both structural and functional work has suggested that there might exist additional allosteric binding sites on these transporters.2 We are interested in developing nonclassical ways of targeting and modulating DAT activity by exploring the idea of employing allosteric modulators of DAT as potential antiaddiction therapeutics. Different from the well-established understanding and availability of selective competitive inhibitors of DAT, the current level of understanding of allosteric modulation of DAT function is very limited. However, convincing evidence of the presence of secondary or allosteric sites in these transporters is provided by the human SERT crystal structure bound with two escitalopram molecules, one in the S1 site and the other in the extra vestibular region.8 Prior to the discovery of the second citalopram molecule in the SERT crystal structure, we pursued the idea of the existence of allosteric sites on DAT and SERT. Through structure/function studies, molecular dynamics and comparative genomics techniques, we identified a unique allosteric domain in SERT located outside the central translocation B


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Figure 2. (A) Radioactive neurotransmitter transport inhibition assay of KM822 against hDAT, hNET, and hSERT in stably transfected MDCK cells. KM822 IC50 for hDAT, hNET, and hSERT is 3.7 ± 0.65, 119 ± 22.82, and 191.6 ± 34.45 μM, respectively. The data was fitted using nonlinear regression, the figure was plotted using average of three independent experiments, and IC50 means and SEM were calculated using the same three experiments. Results are normalized to percent of the highest response in each group. (B) [3H]-DA uptake kinetic assay of wild type DAT transiently transfected COS-7 cells in the absence and presence of 1 μM and 5 μM KM822. Data were fitted to a Michaelis−Menten equation using nonlinear regression. Vmax is 71.5 ± 6.12, 65.8 ± 8.71, and 57.9 ± 4.15 μmol/min/well in the presence of vehicle, 1 μM KM822, and 5 μM KM822, respectively. Km is 6.2 ± 0.69, 7.0 ± 0.58, and 10.54 ± 0.78 μM in the presence of vehicle, 1 μM KM822, and 5 μM KM822, respectively. The figure was generated using an average of four independent experiments. Vmax and Km were calculated based on the same four experiments. For Vmax, vehicle versus 1 μM showed no significance but Vmax for vehicle versus 5 μM showed significant difference (*p < 0.05) when compared using one-way ANOVA with Dunnett’s multiple comparison post-test. No statistical significance was found in Km values using one-way ANOVA with Dunnett’s multiple comparison post-test.

potency in inhibiting DAT-mediated DA reuptake as well as cocaine-associated behaviors in a planarian model of psychostimulant activity.

outward-facing conformation. Key interactions of KM822 at the binding pocket (Figure 1C) includes hydrogen bonding interactions of residues D385 and D476 with nitrogen atoms on the central indole ring of KM822. Other interactions that contribute to the high docking score of KM822 include hydrogen bond interactions between residue T473 and the sulfonamide group, residue D555 and the phenylacetamide group of KM822. The phenylacetamide group has additional interactions with residues R544 and Y548. The central triazinoindole ring also has aromatic cation interactions with residue D476, staggered stacking interaction with residue W84 and aromatic interactions with residue F155 that contribute to its high docking score. KM822 Is a Selective and Noncompetitive DAT Modulator. To characterize the pharmacology and mechanism of action of KM822, dose−response and uptake saturation assays were performed. In dose−response assays against hDAT, hSERT, and the human norepinephrine transporter (hNET), KM822 was found to preferentially inhibit neurotransmitter reuptake in hDAT as compared to hNET and hSERT (Figure 2A). The potency for hDAT transport inhibition is 30 and 50 folds higher than for hNET and SERT, respectively (DAT IC50 = 3.7 ± 0.65 μM, NET IC50 = 119 ± 22.82 μM, and hSERT IC50 = 191.6 ± 34.45 μM). The selective but relatively low potency of KM822mediated inhibition of hDAT could be a desirable feature as many known high potency hDAT inhibitors are known to display addictive properties. KM822 was further characterized for its effect on dopamine uptake kinetics (Figure 2B). KM822 was found to inhibit dopamine uptake in a noncompetitive manner with a decrease in Vmax in the presence of 1 μM (65.8 ± 8.71 μmol/min/well) and 5 μM KM822 (57.9 ± 4.15 μmol/ min/well) compared to vehicle (71.5 ± 6.12 μmol/min/well), with no significant change observed for the apparent affinity for DA (KM) in the absence or presence of KM822 (6.2 ± 0.69, 7.0 ± 0.58, and 10.54 ± 0.78 μM in the presence of vehicle, 1 μM KM822, and 5 μM KM822, respectively). This result suggests a novel allosteric mechanism of action of KM822 acting through a site different from the orthosteric site. Binding Site Characterization of KM822 Confirms the Location of the Allosteric Site Predicted by the in Silico

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RESULTS AND DISCUSSION In Silico Screening Successfully Identifies a Novel DAT Allosteric Modulator. The crystal structure of Drosophila DAT (dDAT) was used to model the outwardfacing conformation of human DAT (hDAT) using the homology modeling program Modeler. Since cocaine binds to the outward-facing conformation of DAT, we rationalized that an allosteric modulator that can inhibit the binding of cocaine should bind to the outward-facing conformation of DAT and hence chose to model the outward-facing conformation using dDAT as a template. Among the 10 hDAT conformations generated by Modeler, the best ranking conformation with the lowest energy was chosen for molecular dynamics (MD) simulations. Analysis of the trajectory from MD simulations from the production run revealed that the allosteric pocket was formed in coordination with the membrane lipids and retained the pocket configuration throughout the simulation (Figure 1A). The allosteric site is in a similar site as previously identified in hSERT.12 The binding pocket is lined by several aromatic and hydropathic residues such as W84, Y88, F155, Y156, F472, and H477 and hydrophilic residues R85, K384, D385, T473, and D476. The fluctuations of residues W84, D385, D476, R544, and Y548 used to define the pharmacophore’s distance restraints were measured during the last 10 ns of the production run of the MD simulation. Screening the in silico libraries for the fivepoint pharmacophore (Figure 1B) resulted in 598 hits that followed a minimum of four out of five pharmacophoric features. Screening the hits against the in silico model for blood-brain barrier (BBB) penetration resulted in 256 hits.14 Docking of the 256 hits to the binding pocket and rank ordering the complexes using customized scoring functions led us to designate compound KM822 as our lead molecule with a high docking score of 87.39. KM822, a substituted triazinoindole molecule (Figure 1C), occupied the entire volume of the allosteric site locking the transporter in an C


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Figure 3. (A) IC50 (mean ± SEM, μM) values of KM822 against the kinetically active DAT mutants. Averages and SEM were calculated from three or more independent experiments. Statistical analysis was performed using Student’s paired t test. The cysteine mutants IC50 values were compared to that of DAT-XC and D476 mutants IC50 were compared to that of DAT-WT. *p < 0.05, **p < 0.01, n.s. no significance. (B) Shows the relative position of each of the mutants with respect to KM822 (shown as orange sticks) in the binding pocket of hDAT represented as gray ribbons.

Figure 4. (A) Immunoblots of biotinylated DAT-XC based cysteine mutants and total DAT protein in the absence and presence of 20 μM KM822. (B) Quantification of the biotinylation data. The biotinylated DAT is normalized to total DAT protein and then KM822-treated biotinylated DAT band intensity is normalized to the untreated band intensity. (C) Representative immunoblots of biotinylated DAT-XC/W84C mutant and total DAT protein in the absence and presence of 20 μM KM822 and 100 μM cocaine. (D) Quantification of the biotinylation data. The biotinylated DAT is normalized to total DAT protein and then KM822-treated biotinylated DAT band intensity is normalized to the untreated band intensity. All the data in the bar graphs B and D represent three or more independent experiments. Statistical analysis was performed using ANOVA with Dunnett’s multiple comparison test comparing to vehicle. **p < 0.01; n.s., no significance.

Studies. To confirm the in silico predicted binding site and further characterize the location and structural determinants of the binding site of KM822 within DAT we employed DA uptake studies and a biotinylation-based biochemical assay. From the molecular model of DAT bound to KM822 and the previous studies of SERT it is clear that the allosteric binding site of KM822 is distinct from the substrate binding (S1) site. As seen in the molecular model (Figure 1) and described above, DAT residues that line this site include W84, R85, Y88, F155, I159, K384, D385, T465, T473, D476, H477, R544, Y548, A550, D555, and I564, where R85 and D476 form a salt bridge that has been proposed to form a gate to the

extracellular space. The DA and cocaine orthosteric binding sites are located beneath this proposed salt bridge in closest proximity to residues F76, V152, and F326.6,15 In order to experimentally validate the KM822-bound DAT molecular model and physically localize the binding site of KM822, we employed structure/function dose−response uptake studies and the substituted cysteine-accessibility method (SCAM).16−18 Several DAT mutants were created in which the amino acids lining the proposed allosteric pocket were systematically replaced by cysteine via site-directed mutagenesis. The only cysteine in the native DAT that is accessible from the D


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and provides a score for the entire residue. According to this calculation, W84 has the least accessibility score of 1.47 when compared to the other residues in the SCAM experiment (Table 1). Because W84 has the least molecular surface

extracellular environment (C306) was replaced with an alanine to create a biotinylation insensitive mutant and this mutant (DAT-XC) was used to generate all the mutants.19 Only DAT mutants with significant expression levels and functional DA transport activity comparable to the WT DAT were progressed further for functional and SCAM studies. For example, mutants F155C, I159C, L474C, H477C, G549C, A550C, and L560C which showed less than 20% maximal DA transport activity (data not shown) as compared to the WT were not included in the functional or the SCAM analyses. As a result, mutants with >50% surface expression levels and >20% DA transport activity in comparison with the WT DAT included in this study were W84C, K384C, D385C, D476E, D476N, T473C, and R544C (Figure 3). In the functional uptake studies using dose− response inhibition assays, KM822’s IC50 values were significantly altered in transiently expressed DAT mutants, W84C, D385C, and D476N, in comparison to WT, suggesting these residues are involved in the binding of KM822 to DAT (Figure 3A). For the W84C mutant we observed an increase in potency and for the D385C and D476N mutants, apparent potency was lowered by the mutation (Figure 3A). It is interesting to note that a previous study20 on a different mutant of W84 to a leucine found a decrease in apparent potency for DAT inhibitors benztropine and GBR12909 and increased apparent potency of cocaine suggesting interactions between this residue and the orthosteric site. To further validate the observations from structure/function studies we performed SCAM studies. For the SCAM studies, the MTSEA-biotin reagent was used to assess accessibility and reactivity of the thiol (-SH) group of the introduced cysteine in the absence or presence of 20 μM KM822. If KM822 interacts with the respective cysteine residue, we hypothesize that it will physically protect the cysteine from being biotinylated. Following biotinylation, biotinylated DAT was affinity-purified using streptavidin beads, separated, and detected using immunoblotting employing an antibody against an HA-tag that is incorporated Nterminally in all the DAT constructs. The total DAT expression in the cells was measured in parallel using unpurified total cell lysates. As expected, the DAT-XC mutant was not biotinylated. We observed that there was no effect of KM822 coincubation on biotinylation of the cysteine mutants D385C, T473C, and R544C (Figure 4). Residue D385 is localized to the extracellular loop (EL) 4, T473 to EL5, and R544 is on the hinge region connecting EL6 which are flexible loop regions, and this could explain why they did not show a significant change in the level of KM822 treated biotinylated mutant DAT as compared to that of the vehicle. This is likely due to fluctuations in the conformation of the flexible loop and consequently the reversibly bound KM822 is unable to completely protect the residues from the covalent attachment of MTSEA-biotin. In case of W84C, its biotinylation is significantly decreased by KM822 coincubation, suggesting KM822 protects it from being biotinylated likely due to a proximal interaction of W84 with KM822 (Figures 3B and 4A, B). Furthermore, we also found that the labeling of this mutant with MTSEA-biotin is not affected by the coincubation of 100 μM cocaine suggesting W84 is not part of the orthosteric S1 site (Figure 4C and D). To further explain these results, we calculated the molecular surface accessibility of each of the investigated residues in the hDAT-KM822 complex. The molecular surface accessibility is calculated using the Connolly algorithm21 using a spherical water molecule of radius 1.4 Å

Table 1. Connolly Surface Accessibility Scores Calculated for Various Residues in the Allosteric Binding Pocket of hDATa residue

accessibility score

W84 R85 Y88 F155 Y156 K384 D385 T465 F472 T473 D476 H477 R544 Y548 A550 D555 I564

1.47 4.59 1.71 1.92 1.81 4.47 3.56 3.95 2.38 12.23 1.56 5.38 7.32 34.92 1.74 1.61 1.94

a

W84 has the least accessible surface score suggesting that it is buried deeper in the pocket while Y548 although lining the pocket is accessible to both lipids and water molecules.

accessibility in our molecular model of KM822 bound within hDAT, this suggests that W84 is the deepest buried residue among the ones examined and the most protected from biotinylation (Figures 1C and 3B). In addition, since cocaine does not protect W84 from covalent attachment of MTSEAbiotin, this suggests that the two compounds bind differently within the transporter: KM822 at the allosteric site and cocaine at the orthosteric S1 site. To further assess the importance of W84 residue in the interaction of KM822 on hDAT, a dose−response experiment was performed. DAT-XC/W84C expressing HEK-293 cells were incubated with varying concentrations of KM822 prior to the addition of MTSEA-biotin. Coincubation with varying KM822 concentration resulted in a dose-dependent decrease in biotinylation signifying an increase in the degree of protection from labeling by increased concentrations of KM822 resulting in an IC50 value of 0.45 ± 0.08 μM (Figure 5). Importantly, the inhibition potency in the biotinylation experiment was similar to the potency of KM822 in inhibiting DA uptake in DAT-XC/W84C transfected COS-7 cells (Figure 3A), further supporting a direct role of W84 in the interaction between KM822 and DAT. In the W84C mutant, cysteine retains the electrostatic interactions with the triazineindole ring of KM822 but with a much less bulky side chain than tryptophan and hence can better accommodate KM822 in the binding pocket which may explain the increase in potency of the W84C mutant for KM822 (Figures 1C and 3B). Also, the comparable potency of KM822 against DATXC/W84C in the DA transport inhibition assay, strongly supports the robustness of our biotinylation assay. These results also indicate that the MTSEA-biotin protection assay against W84C mutant can be used as an E


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Figure 5. (A) Representative immunoblots showing the dose-dependent decrease in biotinylation intensity of DAT-XC/W84C mutant with increasing concentration of KM822 in SCAM analysis using HEK-293 cells. (B) Data in the graph is represented as intensity of biotinylated DATXC/W84C bands normalized to the total DAT-XC/W84C bands for respective concentrations of KM822. The nonlinear regression analysis of the results gave the IC50 of KM822 as 0.45 ± 0.08 μM where average and SEM were calculated from three independent experiments.

mutants. We found that the potency of KM822 remained unchanged in the Y156F-DAT mutant as compared to the WT-DAT but was reduced by 3-fold in the Y335A mutant (Figure 6), suggesting KM822 preferably binds to an outward-

alternative to radioactive competition binding assays to assess the binding affinity of test compounds directly against this novel allosteric binding site. Taken together, the structure/function uptake and SCAM studies supports the notion that KM822 interacts with the proposed allosteric site. It is important to note, that both types of studies are indirect evidence of this. Indirect effects of KM822 binding somewhere else producing long distance effects can as a result not be excluded. Only 3D crystal or cryoEM studies can convincingly demonstrate the exact position of KM822 binding to DAT, and such studies are currently under way with our collaborators. Interestingly, in a recent study on the multihydrophobic substrate transporter MhsT from Bacillus halodurans, which is related to LeuT, it was found that biotinylation of the residue corresponding to W84 was protected by the substrate tryptophan in a similar manner as KM822 protects this site from biotinylation.22 As mentioned above the allosteric site described here shares some structural features with the proposed allosteric S2 site in LeuT and MhsT. Particular it shares the W84 residue in DAT with the corresponding residues in LeuT and MhsT. It could therefore be speculated that the structures mediating the allosteric effects of the substrates in bacterial LeuT and MhsT transporters are conserved in DAT and SERT and can be engaged by other types of compounds in these carriers to produce allosteric effects. Interaction of KM822 with Inward (Y335A) versus Outward (Y156F) Equilibrium Shifting DAT Mutations. To further understand the mechanism of action of KM822, we examined whether KM822 preferentially binds to the outwardor the inward-facing conformation of DAT. We took advantage of two known mutations, i.e., Y156F and Y335A which are commonly used to determine the preferred DAT conformation for various DAT-interacting compounds.23−28 The mutation Y156F removes the interaction of the Y156 hydroxyl group with D79 in the S1 binding site of hDAT which results in an open-outward conformation of DAT. The binding affinity of cocaine and its analogs is unaffected by this mutation, but the potency of atypical DAT inhibitors such as JHW-007 and Smodafinil is significantly reduced.27 On the other hand, the Y335A mutation shifts the DAT conformation equilibrium toward an inward-facing orientation which markedly impairs the potency of cocaine and its analogs but causes only a slight loss in the potency of most benztropine analogs.27 KM822 potency was compared in DA uptake inhibition assay in COS-7 cells transiently expressing WT, Y156F or Y335A DAT

Figure 6. Dose−response assay of KM822 against DAT mutants Y156F and Y335A versus WT-DAT. Nonlinear regression analysis of normalized response gave KM822 IC50 as 3.8 ± 0.88, 3.3 ± 0.45, and 12.8 ± 1.4 μM in WT-DAT, Y156F-DAT, and Y335A-DAT transfected COS-7 cells, respectively. Averages and SEM were calculated from three independent experiments.

facing conformation. To further support this observation, we modeled the inward-facing conformation of WT-DAT using the crystal structure of the inward-facing conformation of LeuT.29 Structural comparison of the outward-facing and inward-facing conformational models of DAT revealed significant conformational changes in the TMDs including hinge motions of several helices leading to an overall root mean squared deviation of ∼9 Å. In the inward-facing conformation, TMD 1, 3, 10, and 11 have significant movement that clearly occlude the formation of the allosteric site (Figure 7A). A study by Cheng and Bahar found similar significant movements of TMDs during the transition from outward- to inward-facing conformations of DAT in their MD simulations.30 We attempted to dock KM822 to the inwardfacing conformation, and as expected the triazineindole ring had steric hindrances from TMD 1, 10 and 11, while EL4, 5, and 6 had no interactions with the rest of the molecule (Figure 7B), suggesting that KM822 does not bind to the inwardfacing conformation of DAT. A caveat to our model is that it is based on the inward-facing conformation of LeuT and no crystal structure of the inward-facing conformation of human DAT is available to validate the model. In addition, there are several differences in the secondary structure between DAT F


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Figure 7. KM822 does not bind to the inward-facing conformation of hDAT. (A) Structural model of inward-facing conformation of hDAT represented as ribbons. Transmembrane domains and loop regions that contributed to the binding pocket of KM822 in the outward-facing conformation are colored as follows: TMD1 = blue, TMD10 = green and TMD11 = pink; EL4 = magenta, EL5 = orange; EL6 = mauve while the rest of the protein is colored gray. KM822 does not bind to the inward-facing conformation and is sterically hindered from entering the binding pocket while the rest of the molecule sits outside the binding pocket. (B) Schematic interaction map of KM822 docked to inward-facing model of hDAT generated using ligand interactions module of MOE. Part of the ligand that is clashing with hDAT is shown in red and the rest of the molecule is outside the binding pocket of hDAT with no specific interactions. Legend details the nature of interactions.

Figure 8. Representative immunoblots of biotinylated (A) DAT-XC/T316C and (B) DAT-XC/T316C and their respective total DAT protein in the absence and presence of 20 μM KM822 and 100 μM cocaine. Bar graphs represent quantification of the biotinylation data for each mutant. The biotinylated DAT for each mutant is normalized to total DAT protein and then KM822-treated biotinylated DAT band intensity is normalized to the untreated band intensity. The data for each mutant represents eight independent experiments. Statistical analysis was performed using ANOVA with Dunnett’s multiple comparison test comparing to vehicle. **p < 0.01; *p < 0.05; n.s., not significant.

and LeuT, e.g., in the length and structure of EL4 which participates in binding of KM822 in DAT. Previous studies by

Shan et al. have demonstrated that the S2 site in LeuT is similar to the S2 site in DAT despite the differences in the G


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Figure 9. (A) Dopamine transport inhibition assay of cocaine in the presence of varying concentrations of KM822 in hDAT transfected COS-7 cells. IC50’s of cocaine are 1.2 ± 0.47 μM (at KM822 = 0), 4.2 ± 0.42 μM (at KM822 = 1 μM), and 8.1 ± 2.62 μM (at KM822 = 5 μM). The figure was plotted using average of three independent experiments, and IC50 means and SEM was calculated using the same three experiments. Results are normalized to percent of the highest response in each group. (B) Dose−response curve for inhibition of dopamine in varying concentrations of cocaine in striatal synaptosomal preparations in the absence and presence of 5 μM KM822. (C) Bar graph shows total DA uptake in striatal synaptosomes which is significantly blocked by 100 μM cocaine and 1 μM GBR12909.

length of EL4 between the two proteins.31 Further, they conclude that binding of substrate at the S2 site likely initiates the conformational changes for transition of an outward or occluded DAT state toward the inward open conformations. However, due to lack of a crystal structure of the inward open conformation of DAT, these observations have not been fully validated. These caveats could limit the validity of our inwardfacing DAT model, but we are encouraged by the fact that the experimental results are in agreement with the prediction of the model. Taken together, these results indicate that KM822 prefers the outward-facing conformation of DAT similar to cocaine and its analogues19,27 but through a mechanism distinct from cocaine as the two compounds occupy different binding sites within DAT. Schmitt et al.32 also reports that another DAT mutant, W84L, shifts the DAT conformation equilibrium toward outward-facing conformation. It is possible that our W84C mutant also adopts a similar outward-open conformation which is preferred by KM822 which could also explain the increase in potency with the W84C mutant. In addition, although the outward-facing conformation is a preferred mode for cocaine and other analogs that have stimulatory effects, there are some DAT inhibitors that are known to prefer outward-facing conformation but produce atypical behavioral effects.33,34 Thus, despite KM822 displaying cocaine-like preference for DAT conformation, its low inhibitory potency with a noncompetitive mechanism of action could make this compound a prototype for another class of atypical DAT inhibitors with an allosteric mechanism of action and with therapeutic potential. KM822 Influences the Conformation of DAT Domains. Previously, the SCAM method has also been used to characterize conformational states in DAT.19 In those studies, the residues T316 and A372 that are present in TMD6 and TMD7, respectively, and face the extracellular side of DAT were used to monitor DAT conformations as their accessibility was shown to be influenced by the binding of cholesterol and

cocaine molecules which are known to promote the outwardfacing conformation of DAT. We used this method to further study the effects of the KM822 interaction with DAT and compared it to that of cocaine. Previously, Hong and Amara19 showed that the DAT-XC/T316C mutant displayed increased labeling with a maleimide-based biotin molecule in the presence of cocaine, while DAT-XC/A372C showed no change in labeling when treated with cocaine. We observed the same results with MTSEA-biotin labeling with DAT-XC/ T316C displaying increased labeling (Figure 8A) and DATXC/A372C showing no change in labeling in the presence of 100 μM cocaine (Figure 8B). When comparing these results with KM822-treated cysteine mutants, a similar effect was observed as the presence of 20 μM KM822 increased the MTSEA-biotin labeling of DAT-XC/T316C DAT while there was no significant change in the labeling of DAT-XC/A372C (Figure 8). These results indicate that KM822 and cocaine both orient the conformation of DAT in comparable ways and, thus, influence the position and accessibility of DAT mutants DAT-XC/T316C and DAT-XC/A372C toward MTSEAbiotin labeling to a similar extent. KM822 Affects Psychostimulant Activity in the in Vitro and ex Vivo Studies. To further study the allosteric effects of KM822, we examined if KM822 would affect cocaine inhibition of DAT by performing a dose−response curve of KM822 on cocaine−DAT interactions in DAT transfected COS-7 cells. As shown in Figure 8, KM822 dose-dependently decreased the potency of cocaine in DA transport inhibition assay. The potency of cocaine in the absence of KM822 was 1.2 ± 0.47 μM (IC50), whereas in the presence of 1 and 5 μM KM822, cocaine’s potency decreased significantly to 4.2 ± 0.42 and 8.1 ± 2.62 μM, respectively (Figure 9A). In addition, KM822 effects were evaluated in ex vivo striatal synaptosomal preparations that contain DAT in its native environment. A similar trend was observed to that of the in vitro cell based assays with KM822 reducing the affinity of cocaine (IC50 is 0.18 ± 0.05 μM in the absence of KM822 versus 0.75 ± 0.31 H


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Figure 10. KM822 decreases cocaine-induced inhibition of DA uptake (app Km) in the NAc. Shown are the mean ± SEM for (A) stimulated DA release expressed as a percent of baseline (BL) and (B) DA uptake inhibition following cumulative concentrations of cocaine. 50 μM KM822 did not affect DA release but significantly reduced the effects of cocaine. Statistical analysis was performed using two-way repeated measures ANOVA and indicated a significant main effect of cocaine on DA uptake inhibition (app Km; F(4,10) = 215.9, p < 0.001), a significant interaction (F(4,40) = 4.13, p < 0.01), but no significant main effect of drug (F(1,10) = 2.75, p = 0.13). Bonferroni analyses indicated that KM822 significantly attenuated the effects of cocaine at the 30 μM cocaine concentration. ***p < 0.001.

Figure 11. Locomotion assay in planarians. (A) KM822 has no effect on baseline locomotion. (B) It blocks locomotion elicited by cocaine and amphetamine but not by nicotine. ***p < 0.001, n.s.; not significant. Statistical analysis was performed using Student’s t test.

μM in the presence of 5 μM KM822) toward DAT in native tissue (Figure 9B). To further examine the effect of KM822 on DA release and uptake in an intact ex vivo model, fast scan cyclic voltammetry was performed on striatal slices containing the Nucleus accumbens. As shown in Figure 10A, KM822 did not affect cocaine-induced changes in DA release. However, KM822 significantly reduced the ability of cocaine at inhibiting the DAT as demonstrated in attenuated changes in apparent Km (app Km) when incubated with cocaine (Figure 10B). In conclusion, we show that KM822 dose-dependently decreased the potency of cocaine in inhibiting the transport of DA by DAT when evaluated in three different assays, i.e., the in vitro dose−response assay of DA transport in DAT-transfected COS-7 cells, the ex vivo assays of DA transport in striatal synaptosomes, as well as in the slice preparations containing DAT in its native environment. It is interesting to note that the effect of KM822 on the interaction of compounds with the orthosteric site differs as we do observe a decrease in apparent affinity for the substrate dopamine in the presence of KM822 but different from what we observe with cocaine this was not significant. This could be explained by the fact that dopamine is a substrate and cocaine is a nontransported inhibitor, but it could also suggest there are subtle differences in the way cocaine and dopamine interacts with the orthosteric site. These results prove the robustness of KM822 in serving as a potential starting point for developing molecules that could interfere with the addictive properties of psychostimulants like cocaine and therefore could have therapeutic potential.

KM822 Suppresses Psychostimulant-Associated Behaviors in a Planarian Model of Addiction. Based on the results on cocaine−DAT interaction, we finally tested KM822 on a cocaine-associated behavior. Demonstrating an activity of KM822 in a behavioral assay further strengthens the idea of KM822 as a promising lead for the development of substance abuse disorder therapeutics. As proof-of-concept, a planarian model of psychostimulant activity35 was employed to examine the effect of KM822 on a psychostimulant-associated behavior. Planaria have been shown to respond to psychostimulants with elevated locomotion and withdrawal symptoms. In addition, these behaviors have been demonstrated to be mediated by neurochemical signaling that is similar to what mediates comparable behaviors in mammalians.35 We found that KM822 specifically blocks the stimulated locomotion elicited by stimulants that interact with DAT (amphetamine and cocaine) resulting in locomotion comparable to vehicle treated animals (Figure 11). On the other hand, KM822 did not block nicotine-elicited locomotion that is not mediated through DAT but the nicotinic acetylcholine receptors. Furthermore, we also demonstrated that KM822 by itself does not affect locomotion in planaria. This nonstimulatory effect of KM822 is desirable as in the past, many DAT-interacting compounds that blocked cocaine’s effects were also shown to be stimulatory by themselves. Our group will further evaluate the nonstimulatory and nonaddicting behaviors of KM822 in other animal models of addiction in future studies. I


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of DAT as it was significantly influenced by its interactions with the lipids from the membrane patch surrounding the pocket and thus maintaining the integrity of the pocket. A five-point receptor pharmacophore was designed using the residues W84, D385, D476, R544, and Y548 which included pharmacophoric features of an aromatic ring, hydrogen bond donor/acceptor pair, and hydrophobic interactions. The five-point pharmacophore was screened against a 3 × 106 compound library in a flexible mode. The hits were then filtered for blood-brain barrier permeability using an in silico model as described previously14 and the resulting hits were docked to the allosteric binding pocket of the hDAT using the genetic algorithm based docking program GOLD (ver 5.2).11,42,43 Docked complexes were ranked using goldscore and reranked using customized scoring scheme.11,43,44 Goldscore is a dimensionless score derived from the energy function that accounts for electrostatic, van der Waals, ligand torsion energy, and flexibility of binding pocket residues.42 Accordingly, the higher the goldscore, the better is the docking fit to the binding pocket; however, since the score approximates the energy terms, it cannot be used to calculate binding affinities or IC50 values. Customized scoring scheme is a knowledge-based scoring function that can be used to increase the weightage for favorable interactions and penalize unfavorable interactions. The method has been successfully used to screen activators of xenobiotic receptors and allosteric activators of the glutamate transporter.43,44 The method involves identifying all the residues within 6 Å from the pharmacophore and scoring the favorable and unfavorable interactions of the ligands with these residues. Stacking interactions with W84, F155, Y88, H477, Y548, W556, H547, and Y551 and hydrogen bonded interactions with D385, D476, K384, D555, R544, T465, and R85 contributed to higher weightage, while unfavorable interactions with opposing chemical features were negatively weighted. Based on the goldscore and customized scoring KM822 ranked the highest among all the ligands and hence was nominated as the lead hit molecule. The docked complex of hDAT and the lead compound KM822 was further subjected to minimization and 1 ns long MD simulation to ensure that the molecule docked stably in the binding pocket. A visual inspection of the simulation showed no significant change in the docked pose or position in the binding pocket which ensured stable docking. To test whether KM822 prefers outward or inward-facing conformation of hDAT, we also modeled hDAT in the inward open conformation using the inward open confrmation crystal structure of LeuT as a template (PDB code: 3TT329) using the Modeler program. The structure was optimized with energy minimization and 3 ns of MD simulations as described above for modeling the outward-facing conformation of hDAT. The resulting hDAT structure in the inward open conformation was used to attempt docking KM822 using GOLD docking program as described above. Chemical Synthesis of KM822. KM822 was obtained from a three-step synthetic scheme by utilizing 5-ethylindoline-2,3-dione and 4-acetamidobenzene-1-sulfonyl chloride as the starting materials. Details of KM822 synthesis and structural characterization are in the Supporting Information. Site-Directed Mutagenesis, Cell Culture, and Transfections. All single and double mutants were generated using the QuikChange (Stratagene, La jolla, CA) site-directed mutagenesis kit using human WT DAT and C306A-DAT as the background, respectively. The mutations were verified by sequencing (Genewiz, LLC). COS-7 and HEK-293 cells were maintained in DMEM (3.5 g/L glucose) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. Stably transfected MDCK cells expressing hDAT, hNET, or hSERT were maintained in DMEM (3.5 g/L glucose) supplemented with 10% FBS and 1% penicillin/streptomycin and blasticidin (5 μg/mL) at 37 °C with 5% CO2. For transient transfections, COS-7 cells were tranfected using the TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI), and HEK-293 cells were transfected using the Lipojet transfection reagent (SignaGen Laboratories, Rockville, MD). Transport Kinetic Assays Using COS-7 Cells. Transport assays were performed as described previously.45 Briefly, COS-7 cells were

CONCLUSION Observations from this and previous studies suggest additional binding sites do exist in DAT and open the door for research into exploring allosteric modulation of conformational changes and function of DAT. We used dynamic modeling and HSB virtual screening of an in silico chemical library to identify a novel allosteric modulator of hDAT. The compound, KM822, modulates the interaction of DAT with the highly addictive psychostimulant cocaine and it prevents the behavioral effects of cocaine in an in vivo model of addiction. The mechanistic studies herein suggest that KM822, similar to cocaine, interacts with an outward-facing conformation of DAT. A systematic evaluation of novel allosteric sites on DAT in the future should significantly advance our understanding of the relationship between the structure and function of this critical therapeutic target and provide valuable insight regarding the range and magnitude of possible modulatory activities. We speculate that molecules interacting with these sites could have therapeutic and clinical potential and that KM822 could serve as a lead for the development of molecules with antiaddictive properties.

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METHODS

Animals and Materials. All experiments using animal subjects were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments involving animal subjects were conducted as preapproved by Drexel University Institutional Animal Care and Use Committees. Radiolabeled substrates, [3H]-dopamine (32.6 Ci/mmol) and [3H]serotonin (23.9 Ci/mmol), were purchased from PerkinElmer (Boston, MA). Cell culture media and supplements, including penicillin/streptomycin, Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM) with glucose, and scintillation fluid, were obtained from Thermo Fisher Scientific (Waltham, MA). Transfection reagents TransIT-LT1 and LipoJet reagent were from Mirus Bio LLC (Madison, WI) and SignaGen Laboratories (Rockville, MD), respectively. Reagents for uptake assays and nonradiolabeled substrates were purchased from SigmaAldrich (St. Louis, MO). MTSEA-biotin was purchased from Biotium, Inc. (Fremont, CA). Reagents for uptake assays and nonradiolabeled substrates were purchased from Sigma-Aldrich (St. Louis, MO). Hybrid Structure-Based Method. The crystal structure of Drosophila melanogaster dopamine transporter (dDAT) (PDB code: 4M48) bound to nortriptyline,36 a tricyclic antidepressant and locked in the outer facing conformation was used for modeling the human DAT (hDAT) using MODELER software package (ver 9.1).37,38 The Modeler program’s output was set to generate 10 low energy conformations, and the best ranking structure was optimized using energy minimization and constrained molecular dynamics simulations with a production run of 10 ns. The final structure from the production run was chosen for further membrane embedded simulations. The modeled hDAT structure was embedded in a POPC membrane patch using the Desmond program (D. E. Shaw Research, New York, NY) with a production run of 600 ns. Optimal positioning of the membrane was computed using the dDAT structures in orientations of proteins in the membrane (OPM) database.39 The results of the simulations were analyzed using inhouse trajectory analysis scripts and visualized using the VMD program.40 Structures from last 10 ns of the simulation was used to screen for cavities using the program VOIDOO41 and the results were rank ordered by the volume of the cavity. The best ranking allosteric cavity chosen was lined by several aromatic residues such as W84, Y88, F155, F472, Y548, W556, and Y551 and by hydrogen bonding residues such as R85, K384, D386, T473, D476, and R544. This pocket was similar to the ATM7 binding pocket that was identified for human SERT previously, thus implicating it as an allosteric site for hDAT.12 The composition of the allosteric binding pocket suggested that the pocket could be specific to the outward-facing conformation J


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rats were anesthetized for 3 min with 2.5% isoflurane before decapitation. Following decapitation, brains were quickly removed and transferred to ice cold oxygenated artificial cerebrospinal fluid (ACSF) containing, (in mM; NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4), pH adjusted to 7.4. Coronal slices containing the NAc were prepared using a vibrating tissue slicer and were then transferred into a continuously oxygenated ACSF bath at room temperature. Following a 30 min recovery period, slices were transferred into a testing chamber flushed with ACSF (32 °C). A bipolar stimulating electrode (Plastics One, Roanoke VA) was placed on the surface of the slice and a carbon fiber microelectrode was placed within the NAc. DA release was elicited with single electrical stimulation (400 μA, 4 ms, monophasic) every 3 min. Baseline DA release and uptake were recorded until stability was achieved across three consecutive stimulations (

Identification of a Novel Allosteric Modulator of the Human Dopamine

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