Tritium-labelled isovaleryl-RYYRIK-NH2 as potential antagonist probe for ORL1 nociceptin receptor

Shogo Inamine, Hirokazu Nishimura, Jinglan Li, Kaname Isozaki, Ayami Matsushima, Tommaso Costa, Yasuyuki Shimohigashi

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2 Citations (Scopus)


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 [3H]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 ± 0.03 nM. From the mutual heterologous binding assays using [3H]isoVa-RYYRIK-NH2 and [3H]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. [3H]isoVa-RYYRIK-NH2 is thus a key tracer to uncover the amino acid residues important for receptor inactivation.

Original languageEnglish
Pages (from-to)5902-5909
Number of pages8
JournalBioorganic and Medicinal Chemistry
Issue number21
Publication statusPublished - Nov 1 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry


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