Interaction of tyrosine 151 in norepinephrine transporter with the 2β group of cocaine analog RTI-113

Abstract

Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100x more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.

Monoamine Transporter Structure, Function, Dynamics, and Drug Discovery: A Computational Perspective

“…Based on docking studies, the Y151F mutation was generated for NET (corresponding to F155 in DAT) and shown to increase the sensitivity of NET to RTI-113 while the reciprocal mutation in DAT (F155Y) decreased sensitivity. Similar to their previous DAT study (28), the authors concluded that cocaine occupies a NET binding site distinct from its S1 substrate site (55). It should be noted, however, that the S1 site was occupied by norepinephrine during the docking experiment.…”

“…Given the similarity (67%) between DAT and NET polypeptide sequences, a LeuT-based DAT model (28,51) was used as a template to construct a NET model. Norepinephrine was found to dock into the S1 site (55). Two distinct binding pockets were identified (Nolan, T.L., unpublished) using a NET model based on the open-to-out LeuT crystal structure 3f3a (56).…”

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

“…We currently lack a complete mechanistic understanding of these properties, as some mutagenesis and comparative modeling studies support the interaction of cocaine at S1, where it could suppress transport by competing with substrate for key interactions (37,54), although other studies support binding near S2, where it could allosterically stabilize an inactive state of the transporter (94 -97). It has been proposed that these binding modes are not mutually exclusive and that cocaine may initially bind near the S2 site before transitioning to S1 following conformational changes (94,95). The current collection of LeuT and dDAT crystal structures supports multiple binding sites for substrates and inhibitors (15-19, 40, 42, 56, 60, 97, 98) and both allosteric and competitive inhibition mechanisms (6, 12, 17-20, 38, 41-43, 55, 60, 97, 99 -101).…”

Behavior of knock-in mice with a cocaine-insensitive dopamine transporter after virogenetic restoration of cocaine sensitivity in the striatum

“…HeLa cells (American Type Culture Collection, Rockville, MD) grown in 96-well plates were transiently transfected with plasmids containing the DAT wt or DAT vcv coding regions as described previously (Hill et al, 2011). …”