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

寻找“更安全”的阿片类药物是最高的临床优先事项，某些国家目前的阿片类药物流行强调了这一点。痛觉肽/孤啡肽FQ肽受体（NOP）是阿片受体家族中第四个也是最不明确的成员。原则上，镇痛可以由所有四种阿片受体介导。鉴于对偏向性激动作用的关注越来越少，多受体靶向已被提出作为一种新的概念，通过使用单一化学实体同时处理多个阿片受体来改善不良副作用。事实上，已经发现了具有双功能活性的新型镇痛药，其靶向微阿片受体（MOP）和NOP，在慢性疼痛检测中比吗啡更有效，但副作用更少。我们最近研究了一系列化学上不同的混合NOP-MOP激动剂诱导的受体磷酸化和G蛋白信号谱，这些激动剂在NOP和MOP上表现出不同的功效。这导致我们假设，具有有限内在活性的双功能NOP-MOP化合物可能是开发具有增加治疗窗口和减少不良反应的新型阿片类药物的有希望的先导结构。本提案将评估G蛋白信号的部分激动作用和激动剂诱导的受体磷酸化的低或缺失的两个参数的组合在多大程度上为有利的镇痛治疗效果提供预测信息，避免滥用责任。我们将使用一组具有不同NOP和mop相关作用平衡的原型工具化合物，包括cebranopadol， AT-034, AT-324， AT-201和AT-121。我们之前已经开发了一组针对NOP和MOP的磷酸特异性抗体，使我们能够评估体内和体外不同的受体激活模式。我们还建立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)评估不同NOP-MOP平衡的典型双功能镇痛药的急性呼吸抑制和增强性能；(2)利用MOP和NOP敲除小鼠对双功能激动剂在急慢性疼痛模型中的镇痛作用进行靶点验证；(3)用表达表位标记受体的敲除蛋白小鼠联合磷酸特异性抗体检测神经性疼痛条件下NOP和MOP的磷酸化；(4)用完全磷酸化缺失的NOP和MOP小鼠评估慢性疼痛模型中NOP和MOP磷酸化对受体反应性的作用。

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.