Characterization of the nociceptin/orphanin FQ peptide receptor (NOP) as a novel pharmacological target for the development of safer analgesics

寻求“更安全”的类阿片是临床上的最高优先事项，某些国家目前类阿片流行突出了这一点。Nociceptin/Nociceptin in FQ肽受体（NOP）是阿片受体家族的第四个并且仍然是特征最少的成员。原则上，镇痛可以由所有四种阿片受体介导。鉴于对偏向激动作用的关注逐渐减少，已经提出多受体靶向作为一种新的概念，通过用单个化学实体同时处理多个阿片受体来改善不利的副作用。事实上，已经发现了具有靶向μ-阿片受体（MOP）和NOP的双功能活性的新型镇痛剂，其在慢性疼痛测定中被证明比吗啡更有效，但副作用更少。我们最近研究了激动剂诱导的受体磷酸化和G蛋白信号传导谱的一系列化学上不同的混合NOP-MOP激动剂，表现出不同的疗效在NOP和MOP。这使我们假设，具有有限内在活性的双功能NOP-MOP化合物可能是有前途的先导结构，用于开发具有增加的治疗窗口和减少的不良反应的新型阿片类药物。本提案将评估G蛋白信号传导中的部分激动和低或不存在激动剂诱导的受体磷酸化的两个参数的组合将在多大程度上为避免滥用倾向的有利镇痛治疗效果提供预测信息。我们将利用一组原型工具化合物，其NOP和MOP相关作用具有不同的平衡，包括西博帕多、AT-034、AT-324、AT-201和AT-121。我们以前已经开发了一组针对NOP和MOP的磷酸位点特异性抗体，这使我们能够评估体内和体外受体活化的不同模式。我们还产生了表达磷酸化缺陷以及表位标记的NOP和MOP受体的新型小鼠模型。利用这些新工具，我们将（1）评估具有不同NOP-MOP平衡的原型双功能镇痛剂的急性呼吸抑制和增强特性;（2）使用MOP和NOP敲除小鼠分析双功能激动剂在急性和慢性疼痛模型中的镇痛作用以进行靶点验证;（3）使用表达表位标记的受体的敲入小鼠，结合磷酸化位点，检测神经性疼痛条件下的NOP和MOP磷酸化。特异性抗体和（4）评估NOP和MOP磷酸化对使用总磷酸化缺陷型NOP和MOP小鼠的慢性疼痛模型中的受体反应性的作用。

The search for “safer” opioids is of highest clinical priority, underscored by the current opioid epidemic in certain countries. The nociceptin/orphanin FQ peptide receptor (NOP) is the fourth and still the least characterized member of the opioid receptor family. In principle, analgesia can be mediated by all four opioid receptors. Given the diminishing focus on biased agonism, multi-receptor targeting has been proposed as a novel concept to ameliorate unfavorable side effects by addressing multiple opioid receptors simultaneously with a single chemical entity. In fact, novel analgesics with bifunctional activity targeting the µ-opioid receptor (MOP) and NOP have been discovered, which proved to be more potent than morphine in chronic pain assays, but exhibiting fewer side effects. We have recently examined agonist-induced receptor phosphorylation and G protein signaling profiles of a series of chemically diverse mixed NOP-MOP agonists, which exhibited variable efficacies at NOP and MOP. This led us to the hypothesis that bifunctional NOP-MOP compounds with limited intrinsic activity might be promising lead structures for the development of novel opioids with increased therapeutic windows and reduced adverse effects. The present proposal will evaluate to what extent a combination of two parameters partial agonism in G protein signaling and low or absent agonist-induced receptor phosphorylation will provide predictive information for favorable analgesic therapeutic effects avoiding abuse liability. We will utilize a set of prototypical tool compounds with different balance of their NOP- and MOP-related effects including cebranopadol, AT-034, AT-324, AT-201 and AT-121. We have previously developed a panel of phosphosite-specific antibodies for NOP and MOP which enables us to assess different patterns of receptor activation in vivo and in vitro. We have also generated novel mouse models expressing phosphorylation-deficient as well as epitope-tagged NOP and MOP receptors. Using these novel tools, we will (1) evaluate acute respiratory depressant and reinforcing properties of prototypical bifunctional analgesics with different NOP-MOP balance; (2) analyse analgesic effects of bifunctional agonists in acute and chronic pain models using MOP and NOP knockout mice for target validation; (3) examine NOP and MOP phosphorylation under conditions of neuropathic pain using knockin mice expressing epitope-tagged receptors in combination with phosphosite-specific antibodies and (4) assess the role of NOP and MOP phosphorylation for receptor responsiveness in chronic pain models using total phosphorylation-deficient NOP and MOP mice.