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

Title
Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site
Authors
Rejwi Acharya DahalAkula Bala PramodBabita SharmaDanielle KroutJames D. Foster차주환Jianjing CaoAmy Hauck NewmanJohn R. LeverRoxanne A. VaughanL. Keith Henry
Keywords
dopamine transporter; serotonin transporter; neurotransmitter sodium symporter; quantitative structure activity relationship; IFD, induced fit docking
Issue Date
2014-10
Publisher
The Journal of biological chemistry
Citation
VOL 289, NO 43, 29712-29727
Abstract
The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.
URI
http://pubs.kist.re.kr/handle/201004/48397
ISSN
00219258
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