title: Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands.
abstract: OBJECTIVE
  The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.
  
  
  METHODS
  Chinese hamster ovary cells stably expressing the human adenosine A(1) receptor and a cyclic 3',5' adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [(3)H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5' (N-ethylcarboxamido) adenosine (NECA).
  
  
  RESULTS
  Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A(1) receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.
  
  
  CONCLUSIONS
  The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.
authors.affiliation: Institute of Cell Signalling, School of Biomedical Sciences, Medical School, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
authors.forename: Jillian G
authors.initials: JG
authors.lastname: Baker
authors.forename: Richard
authors.initials: R
authors.lastname: Middleton
authors.forename: Luke
authors.initials: L
authors.lastname: Adams
authors.forename: Lauren T
authors.initials: LT
authors.lastname: May
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: Barrie
authors.initials: B
authors.lastname: Kellam
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000737:chemistry / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D058851:GPI-Linked Proteins; D017930:Genes, Reporter; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D015336:Molecular Probe Techniques; D015394:Molecular Structure; D011869:Radioligand Assay; D043682:Receptor, Adenosine A1 / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D014162:Transfection; D014970:Xanthines / Q000737:chemistry / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D008024:Ligands; D043682:Receptor, Adenosine A1; D014970:Xanthines; D019830:Adenosine-5'-(N-ethylcarboxamide); C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental