title: Tritium-labelled isovaleryl-RYYRIK-NH2 as potential antagonist probe for ORL1 nociceptin receptor.
abstract: IsoVa-RYYRIK-NH2 is a highly specific antagonist ligand of the opioid receptor-like 1 (ORL1) receptor, an endogenous ligand of which is 17-mer peptide nociceptin. ORL1 antagonists have potential for clinical use as analgesic and antineuropathic drugs, and thus information on the receptor-binding characteristics of antagonists is very important for rational drug design. In the present study, we prepared tritium-labelled isova-RYYRIK-NH2 from its precursor with the 3-methylcrotonyl (CH3)2CCHCO group by a catalytic reduction using tritium gas. The resulting [(3)H]isoVa-RYYRIK-NH2 was evaluated in a saturation binding assay using the COS-7 cell membrane preparations of transiently expressed ORL1. It exhibited more than 90% specific binding with a dissociation constant of 1.21±0.03nM. From the mutual heterologous binding assays using [(3)H]isoVa-RYYRIK-NH2 and [(3)H]nociceptin, isoVa-RYYRIK-NH2 and nociceptin were found to share the receptor-binding site, but each also had a separate specific binding site of its own. They differentiated the two different binding states or conformations of ORL1, which might represent the agonist-active and antagonist-inactive conformations of ORL1. [(3)H]isoVa-RYYRIK-NH2 is thus a key tracer to uncover the amino acid residues important for receptor inactivation.
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Inamine
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Nishimura
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Jinglan
authors.initials: J
authors.lastname: Li
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Kaname
authors.initials: K
authors.lastname: Isozaki
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Ayami
authors.initials: A
authors.lastname: Matsushima
authors.affiliation: Laboratorio di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, Roma, Italy.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Costa
authors.affiliation: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan. Electronic address: shimo@kyudai.jp.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Shimohigashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007700:Kinetics; D009292:Narcotic Antagonists / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D018847:Opioid Peptides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011957:Receptors, Opioid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014316:Tritium / Q000737:chemistry; D000094942:Nociceptin Receptor; D000097629:Nociceptin
chemical_list: D009292:Narcotic Antagonists; D018847:Opioid Peptides; D010455:Peptides; D011957:Receptors, Opioid; D014316:Tritium; D000094942:Nociceptin Receptor; D000097629:Nociceptin
keywords: Antagonist; Nociceptin; Receptor binding assays; Receptor conformations; Receptor probe