Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

Autor:	Dahal RA; From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203., Pramod AB; From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203., Sharma B; From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203., Krout D; From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203., Foster JD; From the Department of Basic Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203., Cha JH; the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224., Cao J; the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224., Newman AH; the Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, Baltimore, Maryland 21224., Lever JR; 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., Vaughan RA; 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., Henry LK; 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.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2014 Oct 24; Vol. 289 (43), pp. 29712-27. Date of Electronic Publication: 2014 Aug 31.
DOI:	10.1074/jbc.M114.571521
Abstrakt:	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. (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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