title: Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands.
abstract: The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds bind with one substrate moiety in the central binding site (the S1 site), whereas the other substrate moiety binds in a distinct binding site (the S2 site). A systematic study of nonconserved SERT/DAT residues surrounding the proposed binding region showed that nonconserved binding site residues do not contribute to selective recognition of substrates in SERT or DAT. This study provides novel insight into the molecular basis for substrate recognition in human transporters and provides an improved foundation for the development of new drugs targeting SERT and DAT.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , DK-2100 Copenhagen, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Andersen
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , DK-8000 Aarhus C, Denmark.
authors.forename: Lucy Kate
authors.initials: LK
authors.lastname: Ladefoged
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , DK-8000 Aarhus C, Denmark.
authors.forename: Trine N Bjerre
authors.initials: TN
authors.lastname: Kristensen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , DK-2100 Copenhagen, Denmark.
authors.forename: Lachlan
authors.initials: L
authors.lastname: Munro
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , DK-8000 Aarhus C, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Grouleff
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , DK-2100 Copenhagen, Denmark.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Stuhr-Hansen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , DK-2100 Copenhagen, Denmark.
authors.forename: Anders S
authors.initials: AS
authors.lastname: Kristensen
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Chemistry, Aarhus University , DK-8000 Aarhus C, Denmark.
authors.forename: Birgit
authors.initials: B
authors.lastname: Schiøtt
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , DK-2100 Copenhagen, Denmark.
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006860:Hydrogen Bonding; D008024:Ligands; D062105:Molecular Docking Simulation; D009154:Mutation; D018377:Neurotransmitter Agents / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008024:Ligands; D018377:Neurotransmitter Agents; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C093171:methanethiosulfonate ethylammonium; D012701:Serotonin; D005020:Ethyl Methanesulfonate; D004298:Dopamine
keywords: Serotonin transporter; alternating access mechanism; dopamine transporter; induced-fit docking; molecular pharmacology; neurotransmitter transport