Pharmacology of Kappa Opioid Receptor

阿片受体（μ、d 和 k）是 Gi/o 偶联的视紫红质样受体。 κ 阿片受体 (KOPR) 激动剂可能有用 作为镇痛剂和止痒剂，没有滥用潜力和与当前使用相关的呼吸抑制 µ 阿片类镇痛药。然而，原型选择性 KOPR 激动剂会引起烦躁或厌恶，这限制了它们的应用 发展。 G 蛋白偶联受体 (GPCR) 通过 G 蛋白或抑制蛋白发出信号以激活不同的下游 效应器。偏向激动剂优先激活 G 蛋白或抑制蛋白介导的信号传导，因此可能具有优势 过度平衡或无偏激动剂，因为它们可以产生副作用较少的治疗效果。然而， 将体外配体偏倚转化为体内药理学尚不确定。纳芙拉芬，唯一选择性 KOPR 激动剂 在临床使用中，日本规定用于治疗尿毒症瘙痒症，在治疗剂量下不会引起烦躁不安。在 在小鼠实验中，我们观察到纳芙拉芬在高于有效剂量的剂量下会引起条件性位置厌恶（CPA） 抗伤害和抗抓伤作用；然而，另外两种选择性 KOPR 激动剂的情况正好相反， U50，488H 和 MOM-SalB。类似地，U50、488H，而不是纳芙拉芬，诱发快感缺乏。因此，我们建立了一种动物 模型以了解抗瘙痒和镇痛作用与烦躁/厌恶分离的机制 KOPR激动剂。重要的是，我们在小鼠大脑中发现 U50、488H 和 MOM-SalB 引起了强烈的 KOPR 磷酸化，但纳芙拉芬没有。此外，U50、488H（但不是纳芙拉芬）增强了某些蛋白质的磷酸化 mTOR 下游和 mTOR 通路可能参与 KOPR 介导的 CPA。对于具体目标 1，我们将 检验激动剂促进 CPA 的能力与其引起 KOPR 磷酸化的能力相关的假设 检查几种结构不同的 KOPR 激动剂。 KOPR 磷酸化将通过免疫印迹检测 使用我们自己的特异性识别磷酸化 KOPR 的抗体。对于具体目标 2，我们将检查 U50、488H 和纳芙拉芬在重要脑区下游磷酸化蛋白质组变化方面的差异 KOPR药理学。此外，我们将研究差异调节蛋白/途径的参与 KOPR 介导的 CPA 和快感缺失。对于特定目标 3，我们将生成突变小鼠品系来检查 KOPR 体内药理学中激动剂促进的 KOPR 磷酸化以及 GRK5 和 GRK6。 KOPR介导 止痒、抗伤害、厌恶和镇静作用以及运动不协调作用将被用作体内 药理措施。我们的“从床边到工作台”的方法，与常用的“从 “从实验室到临床”的策略使我们能够克服将体外基于细胞的结果转化为体内的挑战 药理学。总而言之，拟议的研究将极大地增进我们对 KOPR 药理学的理解 分子、细胞和行为水平以及系统水平的信号传导。确定涉及的信号通路 KOPR 介导的厌恶和快感缺失可能有助于揭示厌恶和抑郁样的潜在机制 一般行为。此外，它可能会导致 KOPR 激动剂的发展，从而降低烦躁不安，并且可以 用作止痒药和镇痛药，这将有助于解决阿片类药物滥用流行的问题。

Opioid receptors (µ, d and k), are Gi/o-coupled, rhodopsin-like receptors. κ opioid receptor (KOPR) agonists may be useful as analgesics and antipruritic agents without abuse potential and respiratory depression associated with currently use µ opioid analgesics. However, prototypic selective KOPR agonists cause dysphoria or aversion, which limits their development. G protein-coupled receptors (GPCRs) signal via both G protein or arrestin to activate different downstream effectors. Biased agonists preferentially activate G protein- or arrestin-mediated signaling and thus may have advantages over balanced or unbiased agonists in that they may produce therapeutic effects with fewer side effects. However, translating in vitro ligand bias to in vivo pharmacology has been uncertain. Nalfurafine, the only selective KOPR agonist in clinical use, is prescribed in Japan for treatment of uremic pruritus, without causing dysphoria at therapeutic doses. In mice, we observed that nalfurafine caused conditioned place aversion (CPA) at doses higher than the effective doses for the antinociceptive and anti-scratch effects; however, the reverse was true for two other selective KOPR agonists, U50,488H and MOM-SalB. Similarly, U50,488H, but not nalfurafine, induced anhedonia. Thus, we established an animal model to understand the mechanisms underlying separation of anti-pruritic and analgesic effects from dysphoria / aversion of KOPR agonists. Importantly, we found in mouse brains U50,488H and MOM-SalB caused robust KOPR phosphorylation, but nalfurafine did not. Also,U50,488H, but not nalfurafine, enhanced phosphorylation of some proteins downstream of mTOR and the mTOR pathway may be involved in KOPR-mediated CPA. For Specific Aim 1, We will test the hypothesis that the ability of agonists to promote CPA is related to its ability cause KOPR phosphorylation by examining several structurally distinct KOPR agonists. KOPR phosphorylation will be detected with immunoblotting using our own antibodies that specifically recognize phosphorylated KOPR. For Specific Aim 2, we will examine the differences between U50,488H and nalfurafine in downstream phosphoproteomic changes in brain regions important in KOPR pharmacology. Furthermore, we will investigate the involvement of differentially regulated proteins / pathways in KOPR-mediated CPA and anhedonia. For Specific Aim 3, we will generate mutant mouse lines to examine the roles of agonist-promoted KOPR phosphorylation and GRK5 and GRK6 in KOPR pharmacology in vivo. KOPR-mediated antipruritic, antinociceptive, aversive and sedative effects and motor incoordination will be used as the in vivo pharmacological measures. Our “from bedside to bench” approach, distinctly different from the commonly used “from bench to bedside” strategy, allows us to circumvent the challenges of translating in vitro cell-based results to in vivo pharmacology. Taken together, the proposed studies will greatly advance our understanding of KOPR pharmacology at the molecular, cellular, and behavioral levels and signaling at a system level. Signaling pathways identified to be involved in KOPR-mediated aversion and anhedonia may shed light on mechanisms underlying aversion- and depression-like behaviors in general. In addition, it may lead to development of KOPR agonists that cause lower dysphoria and can be used as anti-itch medications and analgesics, which will contribute to solving the problems of the opioid abuse epidemic.