title: Synthesis and pharmacological evaluation of [(3)H]HS665, a novel, highly selective radioligand for the kappa opioid receptor.
abstract: Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.
authors.affiliation: †Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Elena
authors.initials: E
authors.lastname: Guerrieri
authors.affiliation: ‡Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, H-6726 Szeged, Hungary.
authors.forename: Jayapal Reddy
authors.initials: JR
authors.lastname: Mallareddy
authors.affiliation: ‡Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, H-6726 Szeged, Hungary.
authors.forename: Géza
authors.initials: G
authors.lastname: Tóth
authors.affiliation: †Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schmidhammer
authors.affiliation: †Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
authors.forename: Mariana
authors.initials: M
authors.lastname: Spetea
mesh_terms: D000700:Analgesics / Q000493:pharmacokinetics; D000701:Analgesics, Opioid / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D044482:Benzeneacetamides / Q000493:pharmacokinetics; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D006801:Humans; D009270:Naloxone / Q000031:analogs & derivatives / Q000494:pharmacology; D010627:Phenethylamines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011759:Pyrrolidines / Q000493:pharmacokinetics; D011869:Radioligand Assay; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: C577967:3-(2-((cyclobutylmethyl)(phenethyl)amino)ethyl)phenol; D000700:Analgesics; D000701:Analgesics, Opioid; D044482:Benzeneacetamides; D010627:Phenethylamines; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D014316:Tritium; D009270:Naloxone; C024224:naloxazone; C045444:U 69593
keywords: binding affinity; radiolabeling; receptor binding assay; selectivity; κ opioid ligand; κ opioid receptor