Neuropilin-1 Is Essential For VEGFA-Mediated Increase Of Sensory Neuron Activity And Development Of Pain-Like Behaviors

Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain via enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with either the small molecule inhibitor EG00229 or the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, using electrophysiology and pain models, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was employed to knockdown NRP-1. NRP-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. NRP-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented VEGFA-induced mechanical allodynia and thermal hyperalgesia in both male and female mice. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system. This study was supported by National Institutes of Health awards (NS098772, NS120663, NS122545, and DA042852 to Rajesh Khanna).

Novel Compounds Targeting Neuropilin Receptor 1 with Potential To Interfere with SARS-CoV-2 Virus Entry.

Neuropilin-1 (NRP-1) is a multifunctional transmembrane receptor for ligands that affect developmental axonal growth and angiogenesis. In addition to a role in cancer, NRP-1 is a reported entry point for several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19). The furin cleavage product of SARS-CoV-2 Spike protein takes advantage of the vascular endothelial growth factor A (VEGF-A) binding site on NRP-1 which accommodates a polybasic stretch ending in a C-terminal arginine. This site has long been a focus of drug discovery efforts for cancer therapeutics. We recently showed that interruption of the VEGF-A/NRP-1 signaling pathway ameliorates neuropathic pain and hypothesize that interference of this pathway by SARS-CoV-2 Spike protein interferes with pain signaling. Here, we report confirmed hits from a small molecule and natural product screen of nearly 0.5 million compounds targeting the VEGF-A binding site on NRP-1. We identified nine chemical series with lead- or drug-like physicochemical properties. Using ELISA, we demonstrate that six compounds disrupt VEGF-A-NRP-1 binding more effectively than EG00229, a known NRP-1 inhibitor. Secondary validation in cells revealed that all tested compounds inhibited VEGF-A triggered VEGFR2 phosphorylation. Further, two compounds displayed robust inhibition of a recombinant vesicular stomatitis virus protein that utilizes the SARS-CoV-2 Spike for entry and fusion. These compounds represent a first step in a renewed effort to develop small molecule inhibitors of the VEGF-A/NRP-1 signaling for the treatment of neuropathic pain and cancer with the added potential of inhibiting SARS-CoV-2 virus entry.