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.

摘要 以蓝色字体标注的修订 冠状病毒病 (COVID-19) 于 2019 年 12 月首次报道，已席卷全球，导致 造成的巨大损失不仅体现在全球范围内造成的死亡人数（约 1,700,000 人），而且还体现在死亡人数上 世界经济体（约 8.8 万亿美元）。因为感染 SARS-CoV-2（COVD-19 的病原体）可以 由于该病毒通过无症状、症状前和有症状的携带者传播，因此很难被遏制。 我们的初步数据表明，SARS-CoV-2 的主要表面抗原 Spike 蛋白具有镇痛作用。 因此，对传播有增无减的解释（截至 2020 年 12 月 21 日，全球约有 7700 万人被感染） 可能是无症状或症状前的个体不会经历作为症状的疼痛和不适 感染的早期预警信号。我们发现 Spike 蛋白与一种称为神经毡蛋白 1 (NRP- 1）允许病毒进入细胞。血管内皮生长因子-A (VEGF-A) – 一种促伤害感受和 血管生成因子，结合 NRP-1，诱导机械性异常性疼痛和热痛觉过敏，但可被 尖峰蛋白。 NRP-1 的药理学拮抗作用也能阻断 VEGF-A 诱导的疼痛行为。这些 结果表明 NRP-1 是一个新的疼痛参与者。 VEGF-A 通过 NRP-1 激活的信号如何导致疼痛是一个研究领域 开放式问题。利用 SARS-CoV-2 Spike 与 NRP 相互作用的这种非典型疼痛缓解功能 1、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 百万种针对 VEGF-A 的化合物（多种小分子和市售天然产物） NRP-1 b1 域上的结合位点。此筛选中排名前 20 的几种“热门”化合物已得到验证 在体外和体内实验中，为我们的计算机预测提供了实验证明。我们已经合作了 与 Regulonix LLC 合作测试 SARS-CoV-2 Spike 蛋白与 NRP-1 结合触发增加的假设 钠和钙通道活性增加伤害感受器活性，最终导致疼痛加剧，这 NRP-1-VEGF-A 相互作用的抑制剂可以阻断信号级联反应。 Regulonix 的具体目标， 以定量目标为指导，包括：(1) 分析 NRP-1 靶向化合物的 (i) 与 NRP- 结合的能力 1; (ii) 使用 ELISA 阻断 NRP-1-VEGF-A 相互作用； (iii) 抑制 VEGF-A 介导的 VEGFR2 的磷酸化是 VEGF-A/NRP-1 信号传导激活的代表。化合物的子集将是 使用全细胞测试了它们抑制感觉神经元中钙和钠电流的能力 电生理学的较小子集正在人类 DRG 中进行测试，以增强严谨性和转化效用 这些化合物； (2) 分析 NRP-1 靶向化合物的体外细胞毒性、物理- 化学、早期 ADME 和 PK 特性，以及对 GPCR、激酶、离子通道和 其他已知的疼痛靶标，包括阿片受体； (3) 表征最佳 2 NRP-1 靶向 化合物在术后疼痛急性模型和神经性神经病的幸存神经损伤（SNI）模型中的作用 疼痛。为了解决安全问题，将在 SNI 模型之前运行运动障碍表型筛查。在最后 在我们的研究中，我们希望能够验证 NRP-1 抑制剂对神经性疼痛的作用。两种化合物将是 在 ALGOGramTM 中进行评估，这是由外部第三方 ANS 提供的一组不同行为疼痛模型 生物技术（里永，法国）。这些实验的完成将表征 NRP-1 作为新型抗- 伤害感受蛋白，将为靶向 NRP-1 治疗持续性神经病理性疼痛提供机会。