Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.

中文翻译：

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小鼠大脑中激动剂特异性信号的磷酸化蛋白质组学方法：mTOR 通路参与 κ 阿片类药物厌恶。

Kappa 阿片受体 (KOR) 激动剂产生镇痛和止痒作用，但其临床应用受到烦躁和幻觉的限制。Nalfurafine 是一种临床使用的 KOR 激动剂，在人类治疗剂量下不会引起烦躁不安或幻觉。我们发现在 CD-1 小鼠中，nalfurafine 产生了剂量依赖性的镇痛和抗划伤作用，就像原型 KOR 激动剂 U50,488H 一样。相比之下，与 U50,488H 不同，纳夫拉芬在有效镇痛和抗抓伤剂量下不会引起厌恶、快感缺乏或镇静作用，也不会引起低水平的运动不协调。因此，我们建立了一个小鼠模型，概括了临床观察的重要方面。然后，我们采用磷酸化蛋白质组学方法来研究不同 KOR 介导效应的潜在机制。一项基于大规模质谱 (MS) 的大脑分析表明，在处理 30 分钟后，纳夫拉芬对磷酸化蛋白质组的扰动与 U50,488H 不同，以大脑区域特异性方式发生。特别是，U50,488H 和 nalfurafine 使在不同脑区的不同细胞成分或信号通路中发现的蛋白质发生磷酸化变化。值得注意的是，我们观察到 U50,488H，而非纳拉芬，激活了纹状体和皮质中的哺乳动物雷帕霉素靶标 (mTOR) 通路。雷帕霉素对 mTOR 通路的抑制消除了 U50、488H 诱导的厌恶，而不影响镇痛、抗划痕和镇静作用以及运动不协调。结果表明 mTOR 通路参与 KOR 激动剂诱导的厌恶。这是首次证明磷酸化蛋白质组学可应用于小鼠大脑中 G 蛋白偶联受体 (GPCR) 的激动剂特异性信号传导，以揭示重要的药理学途径。此外，这是 mTOR 通路介导由 KOR 激活引起的厌恶的前两个报告之一。