Dopamine modulates movement, cognitive, and emotional functions of the brain through activation of dopamine receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Here we present the crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride at 3.15 Å resolution. Docking of R-22, a D3R-selective antagonist to the D3R structure reveals an extracellular extension of the eticlopride binding site that comprises a connected second binding pocket for the aryl amide of R-22.Dopamine is an essential neurotransmitter in the central nervous system and exerts its effects through activation of five distinct dopamine receptor subtypes that belong to the G proteincoupled receptor (GPCR) superfamily. The receptors have been classified into two subfamilies, D1-like and D2-like, on the basis of their sequence and pharmacological similarities (1). The D1-like receptors (D1R and D5R) couple to stimulatory G-protein alpha subunits (G s/olf ), activating adenyl cyclase, whereas D2-like receptors (D2R, D3R and D4R) couple to inhibitory G-protein alpha subunits (G i/o ), inhibiting adenyl cyclase. The high degree of sequence identity (2-3) within the transmembrane helices between D2R and D3R

Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 2+ -binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.Correspondence should be addressed to U.G. (E-mail: gether@sund.ku.dk). Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONST.B. designed and performed the computational experiments, analyzed the data and wrote the manuscript draft together with C.J.L. J.K. generated mutants, carried out pharmacological analyses and contributed to the data analysis. M.L.B. and K.R. generated mutants and carried out pharmacological analyses. L.S. contributed to the computational experiments and manuscript refinement. L.G. participated in the design and performance of the computational experiments. A.H.N. contributed with ideas, benztropine analogues and provided expertise in the pharmacology and medicinal chemistry of DAT inhibitors. J.A.J. contributed with ideas and to the design of experiments and writing of the manuscript. H.W. directed the design and performance of the modeling and computational experiments, participated in data analysis and contributed to writing the manuscript. U.G. supervised the project together with C.J.L., designed experiments, analyzed data and wrote the final manuscript. C.J.L. supervised the project together with U.G., designed experiments, generated mutants, performed pharmacological experiments, analyzed data and wrote the manuscript draft together with T.B.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Neurosci. 2008 July ; 11(7): 780-789. doi:10.1038/nn.2146. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCocaine is an alkaloid derived from the Peruvian Erythroxylon coca plant and has been used as a stimulant for centuries 1 . Today, cocaine is widely abused, especially in the western hemisphere, causing major socioeconomic burdens through increased medical expenses, lost earnings and increased crime 2 . Nonetheless, the molecular mechanisms underlying cocaine's pharmacology and abuse ...

Background (±)-Modafinil has piqued interest as a treatment for ADHD and stimulant dependence. The R-enantiomer of modafinil may have unique pharmacological properties that should be further investigated. Methods (±)-Modafinil and its R-(−)- and S-(+)-enantiomers were synthesized and tested for inhibition of [3H]DA uptake and [3H]WIN 35,428 binding in hDAT WT and mutants with altered conformational equilibria. Data were compared to cocaine and the atypical dopamine uptake inhibitor, JHW 007. R- and S-modafinil were also evaluated in microdialysis studies in the mouse NAc shell and in a cocaine discrimination procedure. Results (±)-, R- and S-Modafinil bind to the DAT and inhibit dopamine uptake less potently than cocaine, with R-modafinil having ~3-fold higher affinity than its S-enantiomer. Molecular docking studies revealed subtle differences in binding modes for the enantiomers. R-modafinil was significantly less potent in the DAT Y156F mutant compared to wild-type DAT, whereas S-modafinil was affected less. Studies with the Y335A DAT mutant showed that the R- and S-enantiomers tolerated the inward facing conformation better than cocaine, which was further supported by MTSET reactivity on the DAT E2C I159C. Microdialysis studies demonstrated that both R- and S-modafinil produced increases in extracellular DA concentrations in the NAc shell less efficaciously than cocaine, and with a longer duration of action. Both enantiomers fully substituted in mice trained to discriminate cocaine from saline. Conclusions R-modafinil displays an in vitro profile different from cocaine. Future trials with R-modafinil as a substitute therapy with the potential benefit of cognitive enhancement for psychostimulant addiction are warranted.

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [ 125 I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on (RS)-CIT recognition (ϳ10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT. SERT,2 like several other members of the SLC6A gene family, acts to remove neurotransmitter from the synapse following neurotransmission (1) and is a major target for treatment of mood disorders, including major depression, anxiety, post-traumatic stress, and obsessive-compulsive disorders (2). Agents that target SERT include tricyclic antidepressants and serotonin-specific reuptake inhibitors (SSRIs) that block 5HT binding and uptake. Despite its clinical significance, very little is understood concerning the structural aspects of SERT and how they relate to its function and antagonist recognition. The current data predict that SERT proteins are composed of 12 transmembrane-spanning segments, intracellular NH 2 and COOH termini and a large second extracellular...