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.

Nonclassical Pharmacology of the Dopamine Transporter: Atypical Inhibitors, Allosteric Modulators, and Partial Substrates

“…By contrast, binding models for the atypical inhibitors benztropine, modafinil and bupropion, as well as the substrates dopamine, amphetamine, and 3,4-methylenedioxy-Nmethylamphetamine reveal a preserved Asp79-Tyr156 bond (Supplemental Fig. 3B), indicating that these ligands do not prevent the DAT from transitioning to an occluded conformation (Bisgaard et al, 2011;Schmitt and Reith, 2011). The fact that atypical inhibitors facilitate a closed-to-out state implies that they affect the transporter conformation in a manner more akin to substrates than to cocaine-like ligands but, unlike true substrates, are not translocated into the intracellular milieu.…”

“…1B) is only nominally impacted (and binding of 3a-benzoyloxytropane is actually increased). This suggests that, unlike cocaine-like ligands, atypical ligands do not require an outward-facing transporter to bind (Kopajtic et al, 2010;Schmitt and Reith, 2011).…”

“…Similarly, binding of cocaine-like compounds was shown to protect DAT transmembrane arginine residues from covalent reaction with phenylglyoxal, whereas benztropine-like compounds failed to impact phenylglyoxal reactivity (Volz et al, 2004). Whole-cell binding studies performed in the presence of Zn 21 (a DAT modulator that loosely binds the extracellular face of the transporter, stabilizing an outward-facing conformation) or in the absence of extracellular Na 1 (which increases the relative number of inward-facing DATs) further hint at specific conformational effects that vary depending on the structure of the bound inhibitor (Loland et al, 2002;Schmitt and Reith, 2011). For example, the affinity of cocaine-like inhibitors for displacement of [ (Fig.…”

“…Molecular modeling studies suggest that cocaine, 2b-carbomethoxy-3b-(4-fluorophenyl)tropane, and methylphenidate promote an outward-facing conformation by breaking a critical hydrogen bond between the side chains of DAT residues Asp79 and Tyr156 (Supplemental Fig. 3A), impeding closure of the extracellular gating network and preventing the transporter from transitioning from an open-to-out state to an occluded state (Beuming et al, 2008;Schmitt and Reith, 2011). By contrast, binding models for the atypical inhibitors benztropine, modafinil and bupropion, as well as the substrates dopamine, amphetamine, and 3,4-methylenedioxy-Nmethylamphetamine reveal a preserved Asp79-Tyr156 bond (Supplemental Fig.…”

“…Recent studies comparing cocaine-like and atypical DAT inhibitors suggest that the behavioral and phenomenological effects of a particular ligand are contingent on how the compound interacts with the transporter. Inhibitors with differing chemical structures exert unique conformational effects on the transporter, stabilizing the protein in different structural states after binding Loland et al, 2008;Schmitt and Reith, 2011), and the nature of these conformational effects can, in turn, influence an inhibitor's rewarding effects Li et al, 2011). The finding that different DAT ligands induce specific conformational changes, which are somehow differentially transduced by the cell, ultimately eliciting distinct downstream effects, suggests the possibility that NSS proteins exhibit some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors (Urban et al, 2007).…”