From the Department of Anesthesiology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China.
Department of Anesthesiology and Critical Care, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.
Anesth Analg. 2020 Sep;131(3):935-942. doi: 10.1213/ANE.0000000000005017.
The misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication.
We determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for β-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison.
The affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the β-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the β-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand.
In conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the β-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.
阿片类药物的滥用部分源于对阿片类药物与阿片受体之间分子相互作用的认识不足。目前仍不清楚为什么有些阿片类药物比其他阿片类药物更容易上瘾。κ 型阿片受体（KOR）在调节疼痛、成瘾和许多其他生理和病理过程中起着关键作用。丁丙诺啡是一种阿片类镇痛药，具有独特的药理学特性，成瘾性较低。在这项研究中，我们研究了丁丙诺啡与 KOR 的相互作用，以深入了解这种药物的安全使用。
我们通过纳洛酮竞争研究确定了丁丙诺啡与 KOR 的结合亲和力。使用在哺乳动物细胞膜中表达的重组 KOR（Chem-1）进行 G 蛋白激活研究，并使用文献中先前描述的稳定转染人 KOR 的人胚肾 293（HEK-293）细胞系进行β-arrestin 研究。使用过表达 KOR 的小鼠神经母细胞瘤 Neuro2A 细胞（N2A-mKOR-tdT）稳定转染 mKOR-tdTomato 融合蛋白研究丁丙诺啡对 KOR 内化的影响。使用全 KOR 激动剂萨维诺林 A 作为对照进行比较。
KOR 对丁丙诺啡的亲和力特征为 Kd 值为 0.1±0.02 nM，比 μ 阿片受体（MOR；2.4±1.2 nM）高约 20 倍。我们的数据表明，丁丙诺啡对 KOR 的作用强于对 MOR。此外，丁丙诺啡在 G 蛋白激活途径中作为 KOR 的部分激动剂，在β-arrestin 募集途径中作为完全激动剂，与萨维诺林 A 相似。KOR 内化进一步证实了β-arrestin 途径的激活。基于计算机的对接模型表明，萨维诺林 A 和丁丙诺啡均与 KOR 完全激动剂 MP1104 共享相同的结合腔。该腔在确定配体的激动剂或拮抗剂效应方面起着重要作用。
总之，丁丙诺啡在 G 蛋白激活途径中是 KOR 的部分激动剂，在β-arrestin 募集途径中是 KOR 的有效激动剂。结构分析提供了对丁丙诺啡与 KOR 相互作用和激活的分子机制的深入了解。
