Modafinil and other compounds—representing different chemical classes of DAT ligands ( Fig. 2 )—were assayed for their ability to inhibit [ 3 H]CFT binding to WT or mutant DATs expressed in whole HEK293 cells. The binding affinities (K i values) of the tested compounds and the observed WT/mutant affinity ratios are listed in Table 1 . Modafinil's binding affinity at WT transporters was relatively low (K i = 2.1 µM); compared to the other reference ligands, modafinil was anywhere from 6- to 100-fold weaker ( Table 1 ). The micromolar level affinity is consistent with prior literature reports of modafinil radioligand binding at the DAT and likely underlies the comparatively high effective dose of modafinil (200–600 mg) in humans [18] , [53] . At the W84L mutant, modafinil showed a significant decrease in affinity (an increase in K i value to 3.8 µM; p<0.05) compared with the WT transporter, resulting in a WT/W84L K i ratio of 0.56 ( Table 1 ). This mutant affinity-shift was strikingly similar to that observed with the atypical ligands benztropine, GBR12909 and bupropion (for each of these ligands, the WT/W84L K i ratio was approximately 0.5). In contrast, the classical DAT inhibitors cocaine, β-CFT and methylphenidate all showed significantly increased binding affinity (decreased K i value) at the W84L mutant: the tropane compounds both gave 3.5-fold improvements, whereas methylphenidate displayed a more modest 2-fold gain. At the D313N mutant, modafinil showed little change in affinity compared with WT (having a WT/D313N K i ratio of 0.95), behaving similarly to bupropion and GBR12909—which gave WT/D313N K i ratios of 0.90 and 1.05, respectively—but not to any of the cocaine-like ligands ( Table 1 ).

Zinc is another important endogenous modulator of the DAT; in vivo, it forms organometallic coordinations with three residues at the top of the extracellular vestibule of the transporter (H193, H375 and E396). By loosely “grasping” these three residues on the external protein face, zinc likely impedes the transition between outward- and inward-facing conformations, biasing the equilibrium in favor of the outward-facing state [41] . Effects of exogenously-applied Zn 2+ are observable experimentally at micromolar concentrations: Zn 2+ increases the binding of β-CFT and cocaine [44] , [45] and can partially overcome the effects of DAT mutations exerting an inward-facing conformational bias (the opposite of the W84L or D313N mutations), such as the Y335A [41] , D345N [57] and W267L [58] mutants. We thus used Zn 2+ to investigate the conformational preference of modafinil and the other DAT ligands. By increasing the population of outward-facing DATs and (at least partially) reversing the effect of extracellular Na + depletion, zinc can highlight compounds that selectively bind to an outward-facing state. Under sodium-free buffer conditions, the addition of 10 µM Zn 2+ significantly increased the binding affinity (decreased the K i value) of cocaine and methylphenidate at WT transporters ( Table 2 ). For inhibition of [ 3 H]CFT binding by cold β-CFT, the presence of Zn 2+ under sodium-free conditions increased the B max value of labeled [ 3 H]CFT by a factor of four, from 125±15.8 fmole/well to 502±78 fmole/well. The calculated absolute K d values for the sodium-free and +10 µM Zn 2+ conditions were not significantly different: 49.32±9.69 and 57.08±6.67, respectively. This zinc-mediated effect—alteration in the B max , but not the K d kinetic parameter—has been demonstrated before in both Na + -free [58] and physionormal Na + (130 mM) buffers [45] , [59] . It is likely that the particular kinetic effects of micromolar Zn 2+ -levels depend on the specific assay protocol and nonlinear curve-fitting algorithm used. Addition of Zn 2+ , however, had little impact on the atypical DAT inhibitors overall (the ratio of K i values obtained in the absence and presence of zinc was close to unity for each compound; Table 2 ). This finding suggests that unlike β-CFT, cocaine or methylphenidate, the interaction of modafinil (like GBR12909, benztropine and bupropion) with the DAT is far less dependent on the transporter assuming an open-to-out conformational state.

Various endogenous ionic species are known affect the conformational equilibrium of the DAT and other NSS-family proteins. For example, recent biophysical studies with LeuT have demonstrated that binding of Na + to the substrate-free (apo) form of the transporter induces a conformational shift toward the open-to-out state, increasing accessibility of the extracellular vestibule [34] and constricting residues near the intracellular gating network [35] , [54] . The sodium gradient present under normal physiological conditions (high extracellular Na + concentration and low intracellular Na + concentration) therefore gives rise to a population of transporters that are predominantly outward-facing, primed to bind ligands approaching from the extracellular milieu [55] . In the absence of significant sodium levels, the transporter effectively shifts between outward and inward-facing conformations [35] . Hence, changing the ionic conditions by removing extracellular sodium (without grossly altering intracellular ionic components) would be expected to increase the preponderance of a “closed-to-out” state amongst the overall population of transporters. Applying this logic to the DAT, we performed intact-cell binding assays with buffer Na + isotonically substituted for the inert and membrane-impermeant cation NMDG + (yielding a functionally 0 mM concentration of extracellular Na + without significantly affecting intracellular ionic conditions), a treatment previously demonstrated to increase the relative number of inward-facing DATs [56] . Replacement of buffer sodium resulted in a decrease of affinity (increase in K i value) for all of the tested DAT inhibitors (compare K i values of WT transporter in Table 1 to those of the Na + -Free condition listed in Table 2 ). However, amongst the inhibitors, modafinil and GBR12909 were least impacted by sodium depletion, displaying 1.4- and 1.8-fold increases in respective K i values.

Adaptive docking of modafinil and other inhibitors in an hDAT model

In an attempt to gain structural insight into the differential interactions of cocaine and modafinil with the DAT, we employed a homology model of the human DAT and docked (R)-modafinil, as well as (S)-bupropion, (d)-methylphenidate and β-CFT with a flexible ligand-adaptive docking procedure. Specific enantiomers of the various DAT inhibitors were used in order to simplify the docking protocol. The (S)-enantiomer of bupropion was selected based upon the stereoselective dopaminergic activity of its primary metabolite (S,S)-hydroxybupropion [60] and the comparatively greater isomeric potency of other (S)-cathinones [61], [62]. Dexmethylphenidate (the threo-(R,R)-isomer of methylphenidate) has been extensively shown to be wholly responsible for the DAT-mediated physiological effects of the racemate [63], [64] and was therefore selected for modeling. The stereochemistry of modafinil differs from other DAT ligands, as modafinil's stereocenter is not the typical asymmetric carbon atom, but a sulfinyl moiety (Fig. 2). Unlike other DAT ligands, which generally possess significant enantioselectivity, (R)- and (S)-modafinil show only mild differences in DAT affinity, with the (R)-enantiomer having marginally greater affinity [65]. In humans, racemic modafinil and (R)-modafinil are active at similar doses, but the (R)-isomer has a more stable pharmacokinetic profile [66] and was recently released to the market as an enantiopure drug (armodafinil); hence, it was selected as the more “active” isomer for docking. β-CFT was chosen over cocaine for its structural rigidity, as flexibility imparted by cocaine's benzoyloxy moiety prevented the docking procedure from converging upon particularly consistent pose clusters. The hDAT model was based upon the structure of LeuT co-crystallized with its substrate leucine, as well as the tricyclic antidepressant desipramine [28]. We previously employed this DAT model in docking of substrates and bivalent substrate-like inhibitors [47]. Two ligand-binding pockets identified in the hDAT model were used for docking—roughly corresponding with the S1 and S2 sites of LeuT—and each inhibitor was docked in both sites. A single candidate was selected from a cluster of top-scoring poses and used as the initial input for further energy minimization of the protein/ligand complex (see Fig. S1 for examples of pose clusters from which potential candidates were selected).

Following docking at the S1 site, modafinil was oriented horizontally (parallel to the plane of the membrane), with the diphenyl ring system facing V152, G153 and Y156 of TM3 and the sulfinylacetamide chain surrounded by F76, A77, D79 of TM1 and F320, S321 and L322 of TM6 (Fig. 3A). In this pose, few strong molecular interactions between modafinil and the DAT were observed, save for hydrogen bonds formed between modafinil's terminal amide nitrogen and residues F76, A77 and D79 (Fig. 4A). At the S2 site, modafinil was positioned just above the extracellular vestibule gating residues R85, F320 and D476 (Fig. 3B); one phenyl ring formed a cation-π interaction with R85 and the protonated amide displayed a combination of hydrogen bonding with D476 and a cation-π interaction with the aromatic side chain of F320 (Fig. 4B). Bupropion docked at a slightly lower position in S1 (Fig. 3C), but like modafinil, the aromatic portion of the molecule was oriented parallel to V152 and enveloped by residues of TM3, whereas the amine nitrogen and bulky tert-butyl group were oriented towards D79, F320 and other adjacent residues of TMs 1 and 6 (Fig. 4C). In the S2 site, while bupropion was positioned marginally higher than modafinil in the extracellular vestibule (Fig. 3D), its strongest molecular interactions—a cation-π interaction with R85 and a hydrogen bond between the amine and D476—were similar (Fig. 4D).

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larger image TIFF original image Download: Figure 3. Final energy-minimized poses of atypical inhibitors docked at the DAT primary (S1) and vestibular (S2) substrate binding sites. Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules (also shown as sticks) are highlighted using gray-colored carbon atoms. The distance between the carboxylate oxygen atom of D79 and the ring hydroxyl moiety of Y156 is displayed in the lower right of each panel (in yellow). (A, B) (R)-modafinil docked at the S1 and S2 sites, respectively—at the S1 site (A), modafinil primarily interacts with D79 and adjacent TM1 residues, whereas at the S2 site (B), it mainly interacts with residues that form the extracellular gating network. (C, D) (S)-bupropion docked at both the S1 (C) and S2 sites (D). Note that for each of the DAT/inhibitor models, the bound inhibitor molecule does not disrupt the D79-Y156 hydrogen bond (i.e. the interatomic distance remains less than 3.5 Å following adaptive docking procedures). https://doi.org/10.1371/journal.pone.0025790.g003

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larger image TIFF original image Download: Figure 4. Molecular interaction diagrams of docked atypical inhibitors. For each panel, the interaction map depicts DAT residues located within 4.5 Å of the bound inhibitor molecule (hydrophobic residues are colored green and polar residues are purple). The most significant (non van der Waals) DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond (green), main-chain hydrogen bond (blue), cation-π bond ( +) or aromatic π-stacking ( ). (A, B) Residue interaction maps for modafinil bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for bupropion bound at the S1 (C) and S2 sites (D), respectively. For both of the atypical inhibitors, binding at the S1 site (panels A and C) gives rise to few strong interactions with the DAT—only their protonated nitrogen atoms form hydrogen bonds—suggesting that recognition of these relatively modest inhibitors (K i >100 nM) is influenced more by molecular shape and steric bulk than by specific polar interactions. https://doi.org/10.1371/journal.pone.0025790.g004

The cocaine-like inhibitors β-CFT and d-methylphenidate also yielded highly populated pose clusters when docked in the S1 and S2 sites (a representative pose cluster for CFT docked at the S1 site is shown in Fig. S1B). At the S1 site, the tropane amine of CFT engaged in hydrogen bonding with D79, with the N-methyl group oriented downward towards F76 and neighboring residues in TMs 1 and 6 (Fig. 5A). The tropane ethylene bridge was directed upward toward the extracellular gate, likely blocking the aromatic side chain of F320 from establishing an interaction with the cationic nitrogen. In addition, the 3β-fluorophenyl ring of CFT participated in π-π stacking aromatic interaction with the side-chain of F326 and the 2β-carbomethoxy moiety formed a hydrogen bond with S422 of TM8 (Figs. 5A and 6A). Many of the interactions and binding pocket residues found for CFT were consonant with those reported in prior molecular simulations of phenyltropane binding at the S1 site (e.g. [31]). In the S2 site, CFT was oriented perpendicular to the plane of the membrane, with the charged tropane amine directed towards the top of the extracellular vestibule (Fig. 5B). Residues from extracellular loop 4 (D385, G386 and P387) helped to shield CFT from the extracellular space, with the backbone of D385 forming a hydrogen bond with the tropane nitrogen (Fig. 6B). The 2β-carbomethyoxy moiety was situated directly adjacent to the side-chains of R85, F155 and D476, but did not disrupt the interaction between R85 and D476. In contrast to the other DAT inhibitors docked in the S2 site, the aromatic portion of CFT dipped below the R85-D476 extracellular gate (Fig. 5B), enabling a π-π stacking interaction between the S1-localized residue Y156 and the 3β-fluorophenyl substituent (Fig. 6B). This binding orientation is relatively consistent with other computational studies modeling cocaine and phenyltropane binding in the extracellular vestibule (S2 site) of the dopamine and noradrenaline transporters in the presence of respective substrates bound at S1 [67], [68].

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larger image TIFF original image Download: Figure 5. Final energy-minimized poses of cocaine-like inhibitors docked at the DAT S1 and S2 sites. Selected binding pocket residues are labeled and rendered as sticks; bound ligand molecules are highlighted using gray-colored carbon atoms. The distances between the oxygen atoms of D79 and Y156 are displayed in the lower right of each panel (in yellow). (A, B) β-CFT docked at the S1 (A) and S2 sites (B); binding of β-CFT at either site disrupts the hydrogen bond between and D79 and Y156 (interatomic distance >3.5 Å), indicating that it promotes an open-to-out conformational state. (C, D) Dexmethylphenidate docked at the respective S1 (C) and S2 sites (D)—similar to CFT, methylphenidate disrupts the D79-Y156 hydrogen bond upon binding at the S1 site (however, at the S2 site, the D79-Y156 interatomic distance is roughly ≈3.6 Å, hence the effect of methylphenidate on the integrity of the hydrogen bond is less conclusive). https://doi.org/10.1371/journal.pone.0025790.g005

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larger image TIFF original image Download: Figure 6. Molecular interaction diagrams of cocaine-like inhibitors docked at the S1 and S2 sites. For each panel, the interaction map depicts DAT residues located within 4.5 Å of the bound inhibitor. As described for Figure 4, the residues are colored based upon their chemical nature and the most significant DAT/inhibitor interactions are labeled with dotted lines and a symbol depicting the chemistry of the interaction formed. (A, B) Residue interaction maps for β-CFT bound at the S1 (A) and S2 sites (B). (C, D) Interaction maps for dexmethylphenidate bound at the S1 (C) and S2 sites (D), respectively. At the S2 site, the interaction pattern of methylphenidate is similar to that of modafinil (compare Figure 6D with Figure 4B). https://doi.org/10.1371/journal.pone.0025790.g006

Despite adopting a slightly different orientation, we found that d-methylphenidate shared many of the same interactions and binding pocket residues with β-CFT when docked at the S1 site (Fig. 5C). In particular, the methyl ester moiety of methylphenidate engaged in hydrogen bonding with the side-chain of S422 and the cationic amine formed a bond with D79 (Fig. 6C). The greatest difference in the binding models of the two inhibitors involved F320: for methylphenidate, the charged piperidine amine group formed both a cation-π interaction with the aromatic side-chain of F320 and a hydrogen bond with the backbone. However, at the S2 site, methylphenidate exhibited an interaction pattern and binding orientation more akin to that of modafinil—forming a cation-π interaction between the ligand aromatic ring and R85, with the protonated ligand amine anchored by a combination of hydrogen bonding with D476 and a cation-π interaction with the aromatic side chain of F320 (Figs. 5D and 6D).

Our in silico modeling data are also consistent with the idea that modafinil interacts with the DAT in a different manner than cocaine-like inhibitors. In a recent study combining molecular simulation and site-directed mutagenesis, Beuming et al. (2008) showed that the presence or absence of a hydrogen bond between D79 and Y156 in a given DAT/ligand complex can provide an indication of the conformational bias engendered by the ligand [31]. The highly conserved TM3 tyrosine residue Y156 interacts with the substrate dopamine as it binds at the S1 site and also participates in the vestibular gating network—consisting of R85, F320 and D476—that partitions the S1 and S2 sites [69], [70]. When dopamine is bound at the S1 site, a hydrogen bond formed between the side chain oxygen atoms of D79 and the hydroxyl moiety of Y156 helps to close the extracellular gate, protecting the S1-bound substrate from infiltration by water from the extracellular space [31]. Hence, the presence of a D79-Y156 hydrogen bond is associated with a “closed-to-out” transporter state. In their molecular dynamics simulations, Beuming et al. (2008) showed that an interatomic distance of less than 3.5 Å (indicative of an intact hydrogen bond) was maintained between the oxygen atoms of D79 and Y156 during binding of DAT substrates (dopamine, amphetamine and MDMA) in the S1 site. In contrast, binding of the classical inhibitors β-CFT and cocaine yielded D79-Y156 distances greater than the 3.5 Å maximum for hydrogen bonding (≈5.5 Å and ≈7.5 Å, respectively), signifying an open vestibular gate in each case. Binding of the atypical inhibitor benztropine, however, resulted in a preserved D79-Y156 hydrogen bond (i.e. an interatomic distance less than 3.5 Å), suggesting that—unlike cocaine—binding of benztropine at the S1 site does not prevent closure of the gate.

In an effort to expand upon this finding, we measured the terminal D79-Y156 distance for each of the modeled DAT inhibitors when bound at either the S1 or the S2 site (Figs. 3 and 4, distance values are indicated in yellow at the bottom of each panel). Modafinil docked at the S1 and S2 sites yielded respective D79-Y156 distances of 2.29 Å and 2.25 Å (Fig. 3A–B), suggesting a preserved hydrogen bond and a closed extracellular gating network. Similarly, the atypical inhibitor bupropion gave respective interatomic distances of 2.34 Å and 2.37 Å when docked at the S1 and S2 sites (Fig. 3C–D). In accordance with the findings of Beuming et al. (2008), docking of β-CFT at the S1 site resulted in a D79-Y156 distance of 4.85 Å, indicative of an open extracellular gate (Fig. 5A). Interestingly, at the S2 site, extension of CFT's 3β-fluorophenyl moiety downward into the S1 site permitted an aromatic stacking interaction with Y156, pushing the tyrosine ring aside and expanding the D79-Y156 distance to 4.94 Å (Fig. 5B). In addition, the classical inhibitor d-methylphenidate also disrupted the D79-Y156 hydrogen bond, yielding S1- and S2-bound distances of 4.12 Å and 3.57 Å, respectively (Fig. 5C–D). This suggests that cocaine-like phenyltropane inhibitors and methylphenidate are capable of inducing an open-to-out transporter conformation upon binding at either the S2 or S1 site.