We modeled and analyzed inhibitors of the benztropine class. These
inhibitors were also suggested to bind in the central cavity, and thus to a
binding site overlapping with that of dopamine. In contrast to cocaine/
CFT, however, they bound in a way that preserved the Asp79-Tyr156
hydrogen bond, and thereby formed a closed binding pocket. Notably,
we have recently assessed conformational changes in DAT promoted by
different benztropine and cocaine analogs by determining reactivity of
a cysteine inserted in position 159, which is believed to be accessible in
the outward facing conformation when the extracellular gate is open
and inaccessible when the gate is closed37. These data showed that,
although cocaine and its analogs probably stabilize the open outwardfacing
conformation, benztropines probably stabilize the closed
conformation. This is consistent with the present binding models
and is supported by simulations of the CFT binding model in a
realistic membrane environment showing water permeating deep
into the CFT-binding site (Fig. 5). Of note, several benztropine
analogs are less effective than cocaine as behavioral stimulants, despite
having similar or higher affinity and selectivity for the DAT than
cocaine36,45. Furthermore, one of the benztropines, JHW 007, was
found to potently antagonize the behavioral effects of cocaine36. It
is tempting, therefore, to speculate that the different binding
configurations between cocaine and benztropines could contribute to
their different pharmacological profiles, and therefore offer structureinformed
hypotheses for molecular mechanisms underlying psychostimulant
action.
In summary, we present here, to the best of our knowledge, the first
comprehensive and experimentally validated model of substrate and
inhibitor binding to the DAT, in the context of the high-resolution
structure of the bacterial transporter homolog LeuT. Our data provide
strong evidence that substrates and inhibitors, including widely abused
psychostimulants such as cocaine and amphetamine, occupy an essentially
similar binding pocket that is buried deep in the DAT molecule
and corresponds to the substrate-binding site in the LeuT structure.
These data therefore finally close the door to the possibility of
generating a competitive inhibitor of cocaine binding that treats
cocaine addiction without itself inhibiting dopamine uptake.
.5 Å) in all trajectories (gray) except for those of CFT and cocaine (black). Error bars are standard deviations of the estimated distances. Dotted lines indicate the minimal distance for hydrogen-bond formation (3.5 Å, horizontal) or an arbitrary fivefold affinity ratio (vertical). Inset, model of the Tyr-Asp interaction seen for the two classes of ligands. (c) Rendering of water permeation into the dopamine- (top) and CFT-binding (bottom) sites. Right, the distribution of water shown over a 25 Å distance along the membrane normal in the region indicated on the left by the rectangular box. The distribution was calculated from the last 5 ns of each of three separate simulations carried out for each ligand-DAT complex (red, blue and black traces, respectively). The locations of the geometric centers of Tyr156 and Asp79 and ligands at the end of the simulations are indicated by blue and green arrows, respectively, and water molecule numbers are indicated as a function of distance. Left, water distribution in context of the structure; silver-colored surfaces indicate the water penetration, with the structures of Tyr156, Asp79 and the ligands rendered.
