title: Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.
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.
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203.
authors.forename: Rejwi Acharya
authors.initials: RA
authors.lastname: Dahal
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203.
authors.forename: Akula Bala
authors.initials: AB
authors.lastname: Pramod
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203.
authors.forename: Babita
authors.initials: B
authors.lastname: Sharma
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203.
authors.forename: Danielle
authors.initials: D
authors.lastname: Krout
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203.
authors.forename: James D
authors.initials: JD
authors.lastname: Foster
authors.affiliation: the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224.
authors.forename: Joo Hwan
authors.initials: JH
authors.lastname: Cha
authors.affiliation: the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224.
authors.forename: Jianjing
authors.initials: J
authors.lastname: Cao
authors.affiliation: the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224.
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.affiliation: the Research Service, Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri 65201, and the Department of Radiology, Radiopharmaceutical Sciences Institute, and Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65211.
authors.forename: John R
authors.initials: JR
authors.lastname: Lever
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203, roxanne.vaughan@med.und.edu.
authors.forename: Roxanne A
authors.initials: RA
authors.lastname: Vaughan
authors.affiliation: From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203, keith.henry@med.und.edu.
authors.forename: L Keith
authors.initials: LK
authors.lastname: Henry
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003042:Cocaine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018374:LLC-PK1 Cells; D008024:Ligands; D008698:Mesylates / Q000378:metabolism; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
chemical_list: D001386:Azides; D050483:Dopamine Plasma Membrane Transport Proteins; D008024:Ligands; D008698:Mesylates; D050505:Mutant Proteins; C076379:RTI 82; C074502:methanethiosulfonate; D003042:Cocaine; D003488:Cyanogen Bromide
keywords: Addiction; Dopamine Transporter; Molecular Docking; Molecular Dynamics; Monoamine Transporter; Photoaffinity Labeling; SCAM; Topane