Genetic and Pharmacological Validation of CRMP2 Phosphorylation as a Novel therapeutic Target for Neuropathic Pain

总结 有效治疗高冲击性疼痛患者是国家疼痛计划的主要目标之一。 战略识别神经病理性疼痛的新靶点和机制将是关键， 开发新的靶向药物，以更好地管理神经性疼痛。我们最近 发现周围神经损伤诱导的轴突导向磷蛋白磷酸化酶的上调 反应介质蛋白2（CRMP 2）和N型电压门控钙（CaV2.2）以及NaV1.7 电压门控钠通道，通过一种未鉴定的 机制在我们的初步研究中，我们发现干扰CRMP 2的磷酸化状态是一种非常有效的方法。 足以保护免受慢性疼痛。其他人发现CRMP 2基因敲入突变小鼠 其中磷酸化位点Ser 522被丙氨酸失活（crmp 2S 522 A）具有降低的灵敏度 痛苦预防CRMP 2磷酸化与对调节异常的影响之间的机制联系 神经性疼痛处理的基础兴奋性突触传递从未被研究过。 是否存在磷酸调节“设定点”，允许CRMP 2在假定的非磷酸调节“设定点”之间“切换”， 磷酸化（非疼痛）和磷酸化（疼痛）状态是未知的。我们发现， pCRMP 2：CRMP 2在人脊髓中变化。根据文献和我们的初步数据，我们 假设损伤诱导磷酸化CRMP 2/CaV2.2和磷酸化CRMP 2/NaV1.7 感觉通路中的上调促进脊髓中的异常兴奋性突触传递， 导致神经性疼痛状态。在目标1中，我们将确定潜在的分子/细胞机制， 在CRMP 2 磷酸化被抑制。在目标2中，我们将尝试直接解决NIH的帮助结束成瘾长期- 长期（HEAL）倡议，以加快科学解决国家阿片类药物公共卫生危机：（a） 确定靶向CRMP 2磷酸化的副作用特征和滥用倾向， 分子，和（B）确定具有小分子的CRMP 2是否可以是阿片样物质节约或减少阿片样物质滥用 责任。我们建议验证CRMP 2磷酸化作为一个新的目标，在神经病理性疼痛， 创新的工具，包括遗传方法（crmp 2S 522 A）小鼠以及非阿片类药物 方法（一种新型CRMP 2-磷酸化靶向化合物）。该应用程序的预期结果是 具有重要的临床意义，因为他们将建立CRMP 2磷酸化作为一种新的疼痛治疗方法 目标通过证明CRMP 2磷酸化的抑制逆转或预防神经性疼痛，我们 将促进新的治疗靶点（CRMP 2磷酸化）的发现和验证， 开发新型疼痛疗法-因此直接解决了RFA-NS-18-043的任务。

SUMMARY Effective treatment of high-impact pain patients is one of the major stated goals of the National Pain Strategy. Identification of new targets and mechanisms underlying neuropathic pain will be critical in developing new target-specific medications for better neuropathic pain management. We recently discovered that peripheral nerve injury-induced upregulation of an axonal guidance phosphoprotein collapsin response mediator protein 2 (CRMP2) and the N-type voltage-gated calcium (CaV2.2) as well as the NaV1.7 voltage-gated sodium channel, correlates with the development of neuropathic pain through an unidentified mechanism. In our preliminary studies, we found that interfering with the phosphorylation status of CRMP2 is sufficient to confer protection from chronic pain. Others have found that a CRMP2 knock-in mutant mouse where the phosphorylation site Ser522 was inactivated with alanine (crmp2S522A) had a decreased sensitivity to pain. A mechanistic link between prevention of CRMP2 phosphorylation and effects on dysregulated excitatory synaptic transmission underlying neuropathic pain processing has never been investigated. Whether there is a phosphoregulatory “set-point” permitting CRMP2 to “toggle” between a presumptive non- phosphorylated (non-pain) and phosphorylated (pain) state is not known. We find that the ratio of pCRMP2:CRMP2 varies in human spinal cords. Based on the literature and our preliminary data, we hypothesize that injury induced phosphorylated-CRMP2/CaV2.2 and phosphorylated-CRMP2/NaV1.7 upregulation in the sensory pathway promotes abnormal excitatory synaptic transmission in spinal cord that leads to neuropathic pain states. In Aim 1, we will determine the molecular/cellular mechanisms underlying aberrant excitatory synaptic transmission and neuropathic pain processing in conditions wherein CRMP2 phosphorylation is inhibited. In Aim 2, we will attempt to directly tackle NIH's Helping to End Addiction Long- term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis by: (a) determining the side-effect profile and abuse liability of targeting CRMP2 phosphorylation with a small molecule, and (b) determine if CRMP2 with a small molecule can be opioid sparing or decrease opioid abuse liability. We propose to validate CRMP2 phosphorylation as a novel target in neuropathic pain using innovative tools that include a genetic approach (crmp2S522A) mice as well as a non-opioid pharmacological approach (a novel CRMP2-phsphorylation targeting compound). The expected results of this application are translationally significant in that they will establish CRMP2-phosphorylation as a novel pain therapeutic target. By demonstrating that inhibition of CRMP2 phosphorylation reverses or prevents neuropathic pain, we will promote the discovery and validation of a novel therapeutic target (CRMP2-phosphorylation) to facilitate the development of novel pain therapeutics – thus directly addressing the mandate of RFA-NS-18-043.