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

Rejwi Acharya Dahal, Akula Bala Pramod, Babita Sharma, Danielle Krout, James D. Foster, Joo Hwan Cha, Jianjing Cao, Amy Hauck Newman, John R. Lever, Roxanne A. Vaughan, L. Keith Henry

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 ([125I]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 [125I]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.

Original languageEnglish (US)
Pages (from-to)29712-29727
Number of pages16
JournalJournal of Biological Chemistry
Volume289
Issue number43
DOIs
StatePublished - Oct 24 2014
Externally publishedYes

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Dopamine Plasma Membrane Transport Proteins
Tropanes
Cocaine
Dopamine
Binding Sites
Ligands
Substrates
Cyanogen Bromide
Schematic diagrams
Carboxylic Acids
Synaptic Transmission
Methionine
Uncertainty
Cysteine
Rats
Catalytic Domain
Esters
Substitution reactions
Molecules
RTI 82

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site. / Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D.; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R.; Vaughan, Roxanne A.; Henry, L. Keith.

In: Journal of Biological Chemistry, Vol. 289, No. 43, 24.10.2014, p. 29712-29727.

Research output: Contribution to journalArticle

Dahal, RA, Pramod, AB, Sharma, B, Krout, D, Foster, JD, Cha, JH, Cao, J, Newman, AH, Lever, JR, Vaughan, RA & Henry, LK 2014, 'Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site', Journal of Biological Chemistry, vol. 289, no. 43, pp. 29712-29727. https://doi.org/10.1074/jbc.M114.571521
Dahal, Rejwi Acharya ; Pramod, Akula Bala ; Sharma, Babita ; Krout, Danielle ; Foster, James D. ; Cha, Joo Hwan ; Cao, Jianjing ; Newman, Amy Hauck ; Lever, John R. ; Vaughan, Roxanne A. ; Henry, L. Keith. / Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 43. pp. 29712-29727.
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