title: Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites.
abstract: γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([(3)H]NCS-382) binding sites. Using a (125)I-labeled photoaffinity derivative of the new GHB ligand, we have performed denaturing protein electrophoresis and detected one major protein band with an apparent mass of 50 kDa from cortical and hippocampal membranes. [(125)I]BnOPh-GHB is the first reported (125)I-labeled GHB radioligand and is a useful tool for in vitro studies of the specific high-affinity GHB binding sites. The related photoaffinity linker [(125)I]4-hydroxy-4-[4-(2-azido-5-iodobenzyloxy)phenyl]butanoate can be used as a probe for isolation of the elusive GHB binding protein.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. pw@farma.ku.dk
authors.forename: Petrine
authors.initials: P
authors.lastname: Wellendorph
authors.forename: Signe
authors.initials: S
authors.lastname: Høg
authors.forename: Paola
authors.initials: P
authors.lastname: Sabbatini
authors.forename: Martin H F
authors.initials: MH
authors.lastname: Pedersen
authors.forename: Lars
authors.initials: L
authors.lastname: Martiny
authors.forename: Gitte M
authors.initials: GM
authors.lastname: Knudsen
authors.forename: Bente
authors.initials: B
authors.lastname: Frølund
authors.forename: Rasmus P
authors.initials: RP
authors.lastname: Clausen
authors.forename: Hans
authors.initials: H
authors.lastname: Bräuner-Osborne
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001567:Benzocycloheptenes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006885:Hydroxybutyrates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008024:Ligands; D015394:Molecular Structure; D010654:Phenylbutyrates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D018080:Receptors, GABA-B / Q000378:metabolism
chemical_list: C553489:4-hydroxy-4-(4-(2-azido-5-iodobenzyloxy)phenyl)butanoic acid; C553488:4-hydroxy-4-(4-(2-iodobenzyloxy)phenyl)butanoic acid; D000345:Affinity Labels; D001386:Azides; D001567:Benzocycloheptenes; D006885:Hydroxybutyrates; D007457:Iodine Radioisotopes; D008024:Ligands; D010654:Phenylbutyrates; D019920:Photoaffinity Labels; D018080:Receptors, GABA-B; C066471:NCS 382; C111420:4-hydroxybutyric acid