The atypical stimulant and nootropic modafinil interacts with the dopamine transporter in a different manner than classical cocaine-like inhibitors

Abstract

Modafinil is a mild psychostimulant with pro-cognitive and antidepressant effects. Unlike many conventional stimulants, modafinil has little appreciable potential for abuse, making it a promising therapeutic agent for cocaine addiction. The chief molecular target of modafinil is the dopamine transporter (DAT); however, the mechanistic details underlying modafinil's unique effects remain unknown. Recent studies suggest that the conformational effects of a given DAT ligand influence the magnitude of the ligand's reinforcing properties. For example, the atypical DAT inhibitors benztropine and GBR12909 do not share cocaine's notorious addictive liability, despite having greater binding affinity. Here, we show that the binding mechanism of modafinil is different than cocaine and similar to other atypical inhibitors. We previously established two mutations (W84L and D313N) that increase the likelihood that the DAT will adopt an outward-facing conformational state--these mutations increase the affinity of cocaine-like inhibitors considerably, but have little or opposite effect on atypical inhibitor binding. Thus, a compound's WT/mutant affinity ratio can indicate whether the compound preferentially interacts with a more outward- or inward-facing conformational state. Modafinil displayed affinity ratios similar to those of benztropine, GBR12909 and bupropion (which lack cocaine-like effects in humans), but far different than those of cocaine, β-CFT or methylphenidate. Whereas treatment with zinc (known to stabilize an outward-facing transporter state) increased the affinity of cocaine and methylphenidate two-fold, it had little or no effect on the binding of modafinil, benztropine, bupropion or GBR12909. Additionally, computational modeling of inhibitor binding indicated that while β-CFT and methylphenidate stabilize an "open-to-out" conformation, binding of either modafinil or bupropion gives rise to a more closed conformation. Our findings highlight a mechanistic difference between modafinil and cocaine-like stimulants and further demonstrate that the conformational effects of a given DAT inhibitor influence its phenomenological effects.

Figure 1. Cartoon representation of the DAT alternating access conformational cycle.

(A) A fully outward-facing conformation with an open extracellular gating network (open-to-out) is established by binding of Na⁺ at the S1 site and is therefore the predominant state in the presence of high extracellular Na⁺ levels and absence of substrate. (B) Following Na⁺ binding, substrate interaction with S1 site residues triggers closure of the extracellular gate, establishing an occluded (closed-to-out) intermediate conformation. (C) Putative interaction of a second molecule of substrate with the vestibular S2 site helps facilitate opening of the intracellular gating network, giving rise to a fully inward-facing (open-to-in) conformation capable of releasing S1-bound substrate and ions into the cytoplasm.

Figure 2. Chemical structures of modafinil and other tested DAT inhibitor ligands.

Atypical inhibitors (top row) exhibited preferential interaction with a more inward-facing transporter conformation, whereas cocaine-like inhibitors (bottom row) preferentially bound to the outward-facing DAT conformation. While modafinil has a chiral sulfoxide moiety, the enantiomers possess little difference in pharmacodynamic activity (hence, only the racemate was tested).

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).

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.

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).

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).