Tritium-Labelled Isovaleryl-RYYRIK-NH2 as Potential Antagonist Probe for ORL1 Nociceptin Receptor
Tritium-Labelled Isovaleryl-RYYRIK-NH2 as Potential Antagonist Probe for ORL1 Nociceptin Receptor
复制标题
氚标记的异戊酰基-RYYRIK-NH2 作为 ORL1 伤害感受素受体的潜在拮抗剂探针
DOI：
10.1016/j.bmc.2014.09.018
复制
发表时间：
2014
影响因子：
3.5
通讯作者：
and Y. Shimohigashi
中科院分区：
文献类型：
--
作者：
S. Inamine;H. Nishimura;J. Li;K. Isozaki;A. Matsushima;T. Costa;and Y. Shimohigashi
IsoVa-RYYRIK-NH2 是阿片受体样 1 (ORL1) 受体的高度特异性拮抗剂配体，其内源性配体是 17 聚肽伤害感受肽。 ORL1拮抗剂具有作为镇痛和抗神经病药物在临床应用的潜力，因此拮抗剂受体结合特性的信息对于合理的药物设计非常重要。在本研究中，我们通过使用氚气催化还原，从其带有3-甲基巴豆酰基(CH3)2C双键CHCO基团的前体制备了氚标记的isova-RYYRIK-NH2。使用瞬时表达的 ORL1 的 COS-7 细胞膜制剂在饱和结合测定中评估所得的 [3H]isoVa-RYYRIK-NH2。它表现出超过 90% 的特异性结合，解离常数为 1.21 ± 0.03 nM。从使用[3H]isoVa-RYYRIK-NH2和[3H]nociceptin的相互异源结合测定中，发现isoVa-RYYRIK-NH2和nociceptin共享受体结合位点，但各自也具有其自己的单独的特异性结合位点。他们区分了 ORL1 的两种不同的结合状态或构象，这可能代表了 ORL1 的激动剂活性和拮抗剂非活性构象。因此，[3H]isoVa-RYYRIK-NH2 是揭示对受体失活重要的氨基酸残基的关键示踪剂。
IsoVa-RYYRIK-NH2is 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-NH2from its precursor with the 3-methylcrotonyl (CH3)2Cdouble bondCHCO group by a catalytic reduction using tritium gas. The resulting [3H]isoVa-RYYRIK-NH2was 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-NH2and [3H]nociceptin, isoVa-RYYRIK-NH2and 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-NH2is thus a key tracer to uncover the amino acid residues important for receptor inactivation.