Targeting the neuropilin-1 receptor (NRP-1)/VEGF-A axis for neuropathic pain

靶向神经毡蛋白-1 受体 (NRP-1)/VEGF-A 轴治疗神经性疼痛

• 批准号: 10321851
• 负责人: Rajesh Khanna
• 金额: $ 41.95万
• 依托单位: REGULONIX, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321851
• 关键词: 2019-nCoV; ACE2; Acute; Address; Afferent Neurons; Affinity; American; Analgesics; Angiogenic Factor; Arizona; Behavioral; Binding; Binding Proteins; Binding Sites; Biological Assay; Biological Sciences; Biotechnology; COVID-19; Calcium; Calcium Channel; Cell Surface Receptors; Cell-Mediated Cytolysis; Cells; Cessation of life; Chemicals; Crystallization; Data; Development; Disclosure; Drug Kinetics; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; France; G-Protein-Coupled Receptors; Goals; Human; In Vitro; Individual; Infection; Ion Channel; KDR gene; Laboratories; Lead; Mediating; Modeling; Molecular; Natural Products; Neurons; Neuropilin-1; Nociception; Nociceptors; Opioid Receptor; Oral; Pain; Pain management; Panthera leo; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Postoperative Pain; Property; Proteins; Proxy; Rattus; Recombinants; Reporting; Role; Route; Running; SARS-CoV-2 antigen; SARS-CoV-2 spike protein; Safety; Series; Signal Transduction; Sodium; Sodium Channel; Specificity; Spinal Cord; Spinal Ganglia; Structure; Surface; Surface Antigens; Surgical incisions; Technology; Testing; Thermal Hyperalgesias; Translating; Universities; VEGFA gene; Validation; Vascular Endothelial Growth Factors; Vesicular stomatitis Indiana virus; Viral; Viral Proteins; Virus; Work; base; biophysical properties; chronic neuropathic pain; chronic pain; coronavirus disease; design; drug candidate; drug discovery; experimental study; in silico; in vivo; inhibitor/antagonist; invention; mechanical allodynia; motor impairment; nerve injury; non-opioid analgesic; novel; novel therapeutics; pain behavior; pain model; pain relief; pain signal; painful neuropathy; pre-clinical; preclinical study; protein protein interaction; receptor; small molecule; small molecule inhibitor; spared nerve; success; uptake; virtual

ABSTRACT Revisions noted in blue font First reported in December of 2019, coronavirus disease (COVID-19) has taken the world by storm, exacting a heavy toll not only in terms of the number of deaths (~1,700,000) it has caused worldwide but also its decimation of world economies (~$8.8 trillion). Because infection with SARS-CoV-2, the causative agent of COVD-19, can be spread by asymptomatic, presymptomatic, and symptomatic carriers, the virus has been difficult to contain. Our preliminary data shows that Spike protein, the major surface antigen of SARS-CoV-2, is analgesic. Therefore, an explanation for the unabated spread (~77 million infected worldwide as of December 21, 2020) may be that asymptomatic or presymptomatic individuals do not experience the pain and discomfort that act as early warning signs of infection. We found that Spike protein binds to a surface receptor called neuropilin 1 (NRP- 1) to allow viral entry into cells. Vascular endothelial growth factor-A (VEGF-A) – a pro-nociceptive and angiogenic factor, binds NRP-1, and induces mechanical allodynia and thermal hyperalgesia that is blocked by Spike protein. Pharmacological antagonism of NRP-1 also blocks VEGF-A–induced pain behaviors. These results identify NRP-1 as a new player in pain. How VEGF-A–activated signaling via NRP-1 leads to pain is an open question. Leveraging this atypical pain-relieving function of the SARS-CoV-2 Spike interaction with NRP- 1, the laboratory of Dr. Rajesh Khanna (University of Arizona (UA)) performed a virtual screen of nearly 0.5 million compounds (diverse small molecules and commercially available natural products) against the VEGF-A binding site on the NRP-1 b1 domain. Several of the top 20 ‘hit’ compounds from this screen have been validated in in vitro and in vivo experiments, providing experimental proof of our in-silico predictions. We have partnered with Regulonix LLC to test the hypothesis that SARS-CoV-2 Spike protein binding to NRP-1 triggers increases in sodium and calcium channel activity to increase nociceptor activity culminating in enhanced pain and that this signaling cascade can be blocked by inhibitors of NRP-1-VEGF-A interaction. Regulonix’s Specific Aims, guided by quantitative goals, are: (1) to profile NRP-1 targeting compounds for their (i) to ability to bind to NRP- 1; (ii) to block the NRP-1-VEGF-A interaction using ELISA; and (iii) to inhibit VEGF-A mediated increase in phosphorylation of VEGFR2, a proxy for activation of VEGF-A/NRP-1 signaling. A subset of compounds will be tested for their ability to inhibit calcium and sodium currents in sensory neurons using whole-cell electrophysiology with a smaller subset being tested in human DRGs to enhance rigor and the translational utility of these compounds; (2) Profile NRP-1 targeting compounds for their in vitro cellular cytotoxicity, physico- chemical, early ADME, and PK properties, and for off-target effects on GPCRs, kinases, ion channels and alternative known pain targets, including opioid receptors; and (3) Characterize the best 2 NRP-1 targeting compounds in an acute model of post-surgical pain and in the spared nerve injury (SNI) model of neuropathic pain. To address safety, a phenotypic screen for motor impairment will be run prior to the SNI model. At the end of our study, we expect to have validated NRP-1 inhibitors for neuropathic pain. Two compounds will be evaluated in the ALGOGramTM, a panel of diverse behavioral pain models by an external third-party – ANS Biotech (Riom, France). Completion of these experiments will characterize the role of NRP-1 as a novel anti- nociceptive protein and will open opportunities for targeting of NRP-1 for persistent neuropathic pain treatment.

蓝色字体指出的摘要修订 冠状病毒病（Covid-19）于2019年12月首次报道，席卷全球 不仅在全球造成的死亡人数（约1,700,000）的数量上，而且也是其决定 世界经济（约8.8万亿美元）。因为COVD-19的病原体SARS-COV-2感染可以 通过无症状，预症状和有症状的载体传播，该病毒很难遏制。 我们的初步数据表明，Spike蛋白是SARS-COV-2的主要表面抗原，是镇痛的。 因此，解释未散布的解释（截至2020年12月21日，全球范围内有7700万个感染） 可能是，渐近或临时性个体并没有遭受痛苦和不适 感染的预警信号。我们发现，尖峰蛋白与称为神经肽1的表面受体结合（NRP- 1）允许病毒进入细胞。血管内皮生长因子A（VEGF-A） - 一种亲人和感人 血管生成因子结合NRP-1，并诱导机械性异常性痛和热痛觉过敏。 尖峰蛋白。 NRP-1的药理拮抗作用还阻止了VEGF-A诱导的疼痛行为。这些 结果将NRP-1识别为疼痛的新玩家。 VEGF-A激活的信号如何通过NRP-1导致疼痛是一种 公开问题。利用SARS-COV-2峰值相互作用与NRP- 1，拉杰什·卡纳（Rajesh Khanna）博士（亚利桑那大学（UA））的实验室进行了近0.5的虚拟屏幕 VEGF-A的百万种化合物（各种小分子和市售天然产品） NRP-1 B1域上的结合位点。该屏幕的前20个“命中”化合物中的几种已得到验证 体外和体内实验，提供了我们的内部预测的实验证明。我们已经合作了 使用Regulonix LLC来检验以下假设：SARS-COV-2峰值蛋白与NRP-1触发者的结合增加 在钠和钙通道活性中，增加了伤害感受器活性，最终导致疼痛增强，这是 NRP-1-1-VEGF-A相互作用的抑制剂可以阻止信号级联反应。 Regulonix的具体目标， 在定量目标的指导下，是：（1）介绍其（i）的NRP-1靶向化合物的能力，以结合NRP- 1; （ii）使用ELISA阻止NRP-1-VEGF-A相互作用； （iii）抑制VEGF-A介导的增加 VEGFR2的磷酸化，这是激活VEGF-A/NRP-1信号传导的代理。一个化合物的子集将是 测试了它们使用全细胞抑制感觉神经元中钙和钠电流的能力 电生理学，在人类DRG中测试了一个较小的子集，以增强严格性和转化实用性 这些化合物； （2）介质NRP-1靶向其体外细胞毒性，物理学的化合物 化学，早期ADME和PK特性，以及针对GPCR，激酶，离子通道和靶点的影响 替代已知疼痛靶标，包括阿片类药物受体； （3）表征最佳的2 NRP-1靶向 急性手术后疼痛和不幸的神经损伤（SNI）模型的化合物 疼痛。为了解决安全性，在SNI模型之前将运行用于运动障碍的表型屏幕。在最后 在我们的研究中，我们预计将验证NRP-1神经性疼痛的抑制剂。两种化合物将是 在AlgogramTM中评估，由外部第三方 - ANS组成的潜水行为疼痛模型。 生物技术（法国RIOM）。这些实验的完成将表征NRP-1作为一种新型抗 - 伤害性蛋白质，并将为NRP-1靶向持续性神经性疼痛治疗的机会。