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

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.NARRATIVE Revisions noted in blue font Regulonix LLC proposes to develop novel small molecules to target a high-affinity protein-protein interaction between neuropilin-1 receptor and (NRP-1) and vascular endothelial growth factor A (VEGF-A). NRP-1 is the surface receptor for the pro-nociceptive factor VEGF-A whose signaling contributes to establishment of chronic neuropathic pain. We leveraged the unique mode of interaction of SARS-CoV-2 Spike protein binding to NRP- 1, in the same pocket as VEGF-A, to discover a novel class of NRP-1 antagonists for chronic pain treatment.