MICA: Defining G protein- and arrestin-dependent signalling pathways of biased DOPr agonists and the relevance to their in vivo effects

最近的一项调查显示，近20%的欧洲人患有持续6个月以上的疼痛。然而，目前治疗慢性疼痛的能力是有限的，因为目前可用的药物不能治愈，也不能提供有限的缓解。骨关节炎和糖尿病神经病变等疾病引起的慢性疼痛会对患者及其家人造成毁灭性的影响，并伴有高抑郁率和睡眠不足。慢性疼痛也构成了一个巨大的经济问题，造成了相当大的工作日损失。因此，为了帮助个体患者和提高生活质量，寻找慢性止痛药是至关重要的。阿片受体（DOPr）是一种神经细胞药物受体，其激活对患者有许多有益的结果，包括缓解慢性疼痛和产生抗抑郁药和减少焦虑的活性。在某些情况下，DOPr激活也可能导致不良影响，包括癫痫行为，这削弱了它们作为药物的潜力。神经元中DOPr的药物激活可能导致大量细胞内信号通路的激活或抑制，从而导致DOPr激活的治疗和/或不良影响。GPCRs上的“偏置”配体的发展是一种新型药物，可以选择性地激活特定的细胞信号传导途径，为开发新型配体开辟了道路，这些配体可能在患者身上产生预期的治疗效果，但同时避免了不良影响。药物研发公司Pharmnovo是该申请的工业合作伙伴，该公司已经制造了许多带有“偏倚”信号的DOPr激活药物，这些药物以不同的方式激活细胞内信号通路。这些药物在慢性疼痛的实验模型中提供疼痛缓解。目前研究的一个问题是，配体的偏向性主要是通过克隆细胞系中过表达的受体来确定的，这可能不能反映生理系统中发生的全局信号反应。因此，确定偏倚和评估其功能意义的最佳方法必须是在生物体或生物体相关部分的内源性受体激活后监测尽可能多的信号输出。因此，如果可以确定DOPr药物在神经元中产生的信号模式（磷酸化蛋白质组），然后通过将这种信号谱与配体产生的功能效应（如诱导疼痛缓解的能力）进行比较，那么应该有可能识别与DOPr药物的理想效果相关的信号通路和与不良效果相关的信号通路。这种方法将有助于设计新的DOPr药物，使其具有最佳的神经元信号特征，从而使患者受益并避免副作用。考虑到这一点，本建议的目标是：-将磷酸化蛋白质组学应用于神经元中的DOPr，比较该受体对偏导信号药物的全局信号反应-将磷酸化蛋白质组学鉴定的神经元信号谱与测试的DOPr激动剂的功能谱（例如它们诱导疼痛缓解的相对能力）相关联，并可能促进癫痫样活动等)——利用这些数据确定最佳的神经元信号谱，用于开发用于治疗疼痛、抑郁和焦虑的DOPr激活药物

A recent survey revealed that almost 20% of the European population suffers pain persisting for more than 6 months. The present ability to treat chronic pain is, however, limited, as the currently available medicines can provide no cure and give limited relief. Chronic pain from diseases such as osteoarthritis and diabetic neuropathy can have a devastating effect on the sufferers and their families, with accompanying high rates of depression and sleep loss. Chronic pain also constitutes a huge economic problem with a considerable loss of working days. Thus, the search for a chronic pain killer is pivotal in order to help individual sufferers and improve quality of life.The delta opioid receptor (DOPr) is a nerve cell drug receptor whose activation leads to a number of beneficial outcomes in the patient including relief of chronic pain and the production of antidepressant and anxiety-reducing activity. DOPr activation may also in some cases lead to undesirable effects including epileptic behaviour, which detracts from their potential as medicines. Drug activation of DOPr in neurons is likely to lead to the activation or inhibition of a large number of intracellular signalling pathways, which contribute to the therapeutic and/or undesirable effects of DOPr activation. The development of "biased" ligands at GPCRs, which are new types of drugs which can selectively activate particular cell signalling pathways, opens the way for novel ligands to be developed that potentially produce the desired therapeutic effects in the patient but which avoid undesirable effects. The drug discovery company Pharmnovo, the industrial partner in this application, has made a number of DOPr activating drugs with "biased" signalling which activate intracellular signalling pathways in different ways. These drugs provide pain relief in experimental models of chronic pain. A problem with current studies is that the bias of a ligand is primarily determined using overexpressed receptors in clonal cell lines, which may not be reflect the global signalling response that occurs in a physiological system. It follows that the best way to determine bias and assess its functional significance must be to monitor as many signalling outputs as possible following activation of the endogenous receptor in the organism or relevant part of an organism. Therefore if the signalling pattern (phosphoproteome) generated by DOPr drugs in a neuron can be determined, then by comparing this signalling profile with functional effects produced by the ligands, such as the ability to induce pain relief, then it should be possible to identify signalling pathways associated with desirable effects and those associated with undesirable effects of DOPr drugs. This approach will assist the design of new DOPr drugs with the best neuronal signalling profile possible to benefit the patient and avoid side effects. With this in mind the objectives of this present proposal are to: - apply phosphoproteomics to DOPr in neurons to compare the global signalling response to biased signalling drugs at this receptor- correlate the neuronal signalling profiles identified from the phosphoproteome with the functional profiles of the tested DOPr agonists (e.g. their relative abilities to induce pain relief, and to potentially promote epileptic-like activity etc)- use these data to identify the very best neuronal signalling profile for the development of DOPr activating drugs for the treatment of pain, depression and anxiety