title: The binding sites for benztropines and dopamine in the dopamine transporter overlap.
abstract: Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational modeling together with site-directed mutagenesis to characterize the binding site for BZTs in DAT. Docking into molecular models based on the structure of the bacterial homolog LeuT supported a BZT binding site that overlaps with the substrate-binding pocket. In agreement, mutations of residues within the pocket, including(2) Val152(3.46) to Ala or Ile, Ser422(8.60) to Ala and Asn157(3.51) to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [(3)H]dopamine uptake inhibition assays and/or [(3)H]CFT competition binding assay. A putative polar interaction of one of the phenyl ring fluorine substituents in JHW007 with Asn157(3.51) was used as a criterion for determining likely binding poses and establish a structural context for the mutagenesis findings. The analysis positioned the other fluorine-substituted phenyl ring of JHW007 in close proximity to Ala479(10.51)/Ala480(10.52) in transmembrane segment (TM) 10. The lack of such an interaction for BZT led to a more tilted orientation, as compared to JHW007, bringing one of the phenyl rings even closer to Ala479(10.51)/Ala480(10.52). Mutation of Ala479(10.51) and Ala480(10.52) to valines supported these predictions with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine.
authors.affiliation: Molecular Neuropharmacology Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Heidi
authors.initials: H
authors.lastname: Bisgaard
authors.forename: M Andreas B
authors.initials: MA
authors.lastname: Larsen
authors.forename: Sonia
authors.initials: S
authors.lastname: Mazier
authors.forename: Thijs
authors.initials: T
authors.lastname: Beuming
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Claus J
authors.initials: CJ
authors.lastname: Loland
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000818:Animals; D001590:Benztropine / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003042:Cocaine / Q000378:metabolism / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D013329:Structure-Activity Relationship
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D001590:Benztropine; D003042:Cocaine; D004298:Dopamine
references.citation: J Pharmacol Exp Ther. 2004 May;309(2):650-60
references.pmid: 14755006
references.citation: Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1683-8
references.pmid: 11818545
references.citation: Eur J Pharmacol. 2003 Oct 31;479(1-3):71-82
references.pmid: 14612139
references.citation: Nat Chem Biol. 2010 Feb;6(2):109-16
references.pmid: 20081826
references.citation: Nature. 2007 Aug 23;448(7156):952-6
references.pmid: 17687333
references.citation: Eur J Pharmacol. 2003 May 9;468(2):109-19
references.pmid: 12742518
references.citation: Science. 2008 Dec 12;322(5908):1655-61
references.pmid: 19074341
references.citation: Nature. 2005 Sep 8;437(7056):215-23
references.pmid: 16041361
references.citation: Mol Pharmacol. 2002 Apr;61(4):885-91
references.pmid: 11901228
references.citation: Nat Neurosci. 2008 Jul;11(7):780-9
references.pmid: 18568020
references.citation: Nat Struct Mol Biol. 2009 Jun;16(6):652-7
references.pmid: 19430461
references.citation: J Pharmacol Exp Ther. 2003 Nov;307(2):801-8
references.pmid: 12966155
references.citation: J Med Chem. 2006 Jan 26;49(2):534-53
references.pmid: 16420040
references.citation: J Biol Chem. 2008 Jun 20;283(25):17691-701
references.pmid: 18426798
references.citation: Eur J Pharmacol. 2003 Oct 31;479(1-3):93-106
references.pmid: 14612141
references.citation: Nature. 2009 May 21;459(7245):347-55
references.pmid: 19458710
references.citation: Pharm Res. 2005 Apr;22(4):603-12
references.pmid: 15846468
references.citation: Pflugers Arch. 2004 Feb;447(5):519-31
references.pmid: 12719981
references.citation: J Neurosci. 2005 Feb 23;25(8):1889-93
references.pmid: 15728828
references.citation: J Phys Chem B. 2009 Nov 12;113(45):15057-66
references.pmid: 19831380
references.citation: Trends Pharmacol Sci. 2006 Jul;27(7):375-83
references.pmid: 16762425
references.citation: J Biol Chem. 2006 Jan 27;281(4):2012-23
references.pmid: 16272152
references.citation: J Biol Chem. 1999 Dec 24;274(52):36928-34
references.pmid: 10601246
references.citation: J Biol Chem. 2010 Mar 12;285(11):8363-74
references.pmid: 19948720
references.citation: Science. 2007 Sep 7;317(5843):1390-3
references.pmid: 17690258
references.citation: Synapse. 2005 Nov;58(2):72-94
references.pmid: 16088952
references.citation: Eur J Pharmacol. 2003 Oct 31;479(1-3):3-12
references.pmid: 14612133
references.citation: Mol Pharmacol. 2006 Nov;70(5):1630-42
references.pmid: 16880288
references.citation: J Biol Chem. 1997 Nov 7;272(45):28321-7
references.pmid: 9353288
references.citation: Med Res Rev. 2002 Sep;22(5):429-64
references.pmid: 12210554
references.citation: J Am Chem Soc. 2010 Feb 3;132(4):1311-22
references.pmid: 20055463
references.citation: Mol Pharmacol. 2008 Mar;73(3):813-23
references.pmid: 17978168
references.citation: Curr Opin Neurobiol. 2007 Jun;17(3):304-12
references.pmid: 17509873
references.citation: J Med Chem. 1997 Dec 19;40(26):4329-39
references.pmid: 9435902
references.citation: J Biol Chem. 2004 Jan 30;279(5):3228-38
references.pmid: 14597628
references.citation: Annu Rev Pharmacol Toxicol. 2003;43:261-84
references.pmid: 12359863
references.citation: J Med Chem. 2004 Jan 15;47(2):337-44
references.pmid: 14711306
references.citation: J Pharmacol Exp Ther. 2009 May;329(2):677-86
references.pmid: 19228996
references.citation: Br J Pharmacol. 2006 Jan;147 Suppl 1:S82-8
references.pmid: 16402124
references.citation: J Pharm Sci. 2008 May;97(5):1993-2007
references.pmid: 17879975
references.citation: J Biol Chem. 2009 Apr 10;284(15):10276-84
references.pmid: 19213730
references.citation: J Med Chem. 1994 Jul 22;37(15):2258-61
references.pmid: 8057273
references.citation: Mol Pharmacol. 2009 Mar;75(3):514-24
references.pmid: 19098122
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9333-8
references.pmid: 16754872
references.citation: Nature. 2007 Oct 11;449(7163):726-30
references.pmid: 17704762
references.citation: Mol Cell. 2008 Jun 20;30(6):667-77
references.pmid: 18570870
references.citation: J Biol Chem. 2010 Jan 15;285(3):2051-63
references.pmid: 19892699