title: Insights into the Modulation of Dopamine Transporter Function by Amphetamine, Orphenadrine, and Cocaine Binding.
abstract: Human dopamine (DA) transporter (hDAT) regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing (OF) and inward-facing states of DAT. hDAT is a target for addictive drugs, such as cocaine, amphetamine (AMPH), and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH), an anticholinergic agent and anti-Parkinson drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT-drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations, and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH, and cocaine and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition toward a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the OF open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine.
authors.affiliation: Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA.
authors.forename: Mary Hongying
authors.initials: MH
authors.lastname: Cheng
authors.affiliation: Department of Cell Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA.
authors.forename: Ethan
authors.initials: E
authors.lastname: Block
authors.affiliation: Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA ; Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University , Beijing , China.
authors.forename: Feizhuo
authors.initials: F
authors.lastname: Hu
authors.affiliation: Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA.
authors.forename: Murat Can
authors.initials: MC
authors.lastname: Cobanoglu
authors.affiliation: Department of Cell Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
authors.affiliation: Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh , Pittsburgh, PA , USA.
authors.forename: Ivet
authors.initials: I
authors.lastname: Bahar
keywords: amphetamine; drug modulation mechanism; human dopamine transporter; orphenadrine cocaine; repurposable drugs