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阿片受体激动剂可能是有用的 作为镇痛剂和止痒剂，不存在滥用潜力和与当前使用相关的呼吸抑制 阿片类镇痛剂。然而，典型的选择性KOPR激动剂会引起焦虑症或厌恶，这限制了他们的 发展。G蛋白偶联受体(GPCRs)通过G蛋白或arrestin来激活不同的下游信号 效应器。偏置激动剂优先激活G蛋白或arrestin介导的信号，因此可能具有优势 超过平衡或无偏向的激动剂，因为它们可以产生副作用较少的治疗效果。然而， 将体外配基偏向转化为体内药理学一直是不确定的。唯一的选择性KOPR激动剂--纳夫拉芬 在临床上使用，在日本用于治疗尿毒症瘙痒，在治疗剂量下不会引起烦躁不安。在……里面 在小鼠身上，我们观察到，在高于有效剂量的条件位置厌恶(CPA)中， 抗伤害性和抗抓挠作用；然而，对于另外两种选择性KOPR激动剂则相反， U50,488H和MoM-Salb。同样，U50,488H可引起快感减退，但不能引起纳呋芬。因此，我们建立了一种动物 从烦躁/厌恶中分离止痒和止痛作用机制的模型 KOPR激动剂。重要的是，我们发现在小鼠的大脑中，U50,488H和MoM-Salb引起了强大的KOPR 磷酸化，但呋喃呋喃不起作用。此外，U50,488H可促进某些蛋白质的磷酸化，但不能促进其磷酸化 MTOR下游和mTOR通路可能参与了KOPR介导的CPA。为实现具体目标1，我们将 检验激动剂促进CPA的能力与其通过以下方式引起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的磷酸化将用免疫印迹法检测 使用我们自己的抗体来识别磷酸化的KOPR。对于特定的目标2，我们将研究 U50、488H与呋喃西林对脑下游重要脑区磷蛋白质组变化的差异 KOPR药理学。此外，我们还将研究差异调控蛋白/通路在 KOPR介导的CPA和快感缺失。对于特定的目标3，我们将产生突变的小鼠品系来研究 激动剂在体内促进KOPR磷酸化和GRK5、GRK6的药理作用。KOPR-中介 止痒、止痛、镇静和镇静作用以及运动不协调将被用作体内的 药理措施。我们的“从床边到板凳”的方法与通常使用的“从床到凳”截然不同。 工作台到床边“策略，允许我们绕过将基于体外细胞的结果转化为体内结果的挑战 药理学。综上所述，建议的研究将极大地促进我们对KOPR药理学的了解 分子、细胞和行为水平以及系统水平上的信号。已确定参与的信号通路 在KOPR中，厌恶和快感缺乏可能有助于揭示厌恶和类似抑郁的潜在机制 一般的行为。此外，它可能导致KOPR激动剂的开发，这些激动剂导致较低的焦虑感，并可以 用作止痒药物和镇痛剂，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.