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

用蓝色字体标注的修订 2019年12月首次报道，冠状病毒病（COVID-19）席卷全球， 严重的死亡人数不仅在世界范围内造成的死亡人数（约1，700，000人）方面，而且在其毁灭性方面也是如此。 世界经济总量（约8.8万亿美元）。因为感染SARS-CoV-2（COVD-19的病原体）， 由于该病毒可由无症状、症状前和有症状的携带者传播，因此难以控制。 我们的初步数据表明，刺突蛋白，SARS-CoV-2的主要表面抗原，是镇痛。 因此，解释了这种有增无减的传播（截至2020年12月21日，全球约有7700万人感染） 可能是无症状或症状前个体不经历疼痛和不适， 感染的早期预警信号。我们发现Spike蛋白与一种称为神经纤毛蛋白1（NRP-1）的表面受体结合。 1)让病毒进入细胞血管内皮生长因子-A（VEGF-A）-一种促伤害性和 血管生成因子，结合NRP-1，并诱导机械性异常性疼痛和热痛觉过敏，其被 刺突蛋白。NRP-1的药理学拮抗作用也阻断VEGF-A诱导的疼痛行为。这些 结果表明NRP-1是疼痛的新参与者。VEGF-A通过NRP-1激活的信号传导如何导致疼痛是一个研究热点。 开放的问题。利用SARS-CoV-2尖峰与NRP相互作用的这种非典型疼痛缓解功能， 1，Rajesh卡纳博士（亚利桑那大学（UA））的实验室进行了近0.5的虚拟屏幕 100万种化合物（各种小分子和市售天然产物） NRP-1 b1结构域上的结合位点。该筛选的前20个“热门”化合物中有几个已经过验证 在体外和体内实验中，为我们的计算机预测提供了实验证据。我们合作了 与Regulonix LLC合作，以检验SARS-CoV-2刺突蛋白与NRP-1触发物结合增加的假设。 在钠和钙通道活性中增加伤害感受器活性，最终增强疼痛， 信号级联可被NRP-1-VEGF-A相互作用的抑制剂阻断。Regulonix的具体目标， 由定量目标指导的是：（1）分析NRP-1靶向化合物的（i）结合NRP-1的能力， 1;（ii）使用ELISA阻断NRP-1-VEGF-A相互作用;和（iii）抑制VEGF-A介导的NRP-1-VEGF-A相互作用的增加。 VEGFR 2的磷酸化是VEGF-A/NRP-1信号传导激活的代理。化合物的子集将是 使用全细胞测试它们抑制感觉神经元中的钙和钠电流的能力 电生理学，在人类DRG中测试较小的子集，以增强严谨性和平移效用 （2）分析NRP-1靶向化合物的体外细胞毒性、生理毒性和细胞毒性。 化学、早期ADME和PK特性，以及对GPCR、激酶、离子通道和 替代已知的疼痛靶点，包括阿片受体;和（3）表征最佳的2个NRP-1靶向 在手术后疼痛的急性模型和神经病理性疼痛的备用神经损伤（SNI）模型中， 痛苦为了解决安全性问题，将在SNI模型之前进行运动障碍的表型筛选。年底 在我们的研究中，我们期望已经验证了NRP-1抑制剂对神经性疼痛的作用。两种化合物将 在ALGOGramTM中进行评估，ALGOGramTM是由外部第三方ANS提供的一组不同的行为疼痛模型。 Biotech（Riom，法国）。这些实验的完成将表征NRP-1作为一种新的抗肿瘤药物的作用。 伤害感受蛋白，并且将为靶向NRP-1用于持续性神经性疼痛治疗打开机会。

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.