Small molecules directly targeting the voltage-gated sodium channel (VGSC) Na _V 1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the Na _V 1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked Na _V 1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to Na _V 1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the Na _V 1.7 CRS over other Na _V isoforms. Substitution of the Na _V 1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased Na _V 1.7 currents. A cell-penetrant decoy peptide corresponding to the Na _V 1.7-CRS reduced Na _V 1.7 currents and trafficking, decreased presynaptic Na _V 1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the Na _V 1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a Na _V 1.7 -targeted gene therapy, we packaged a plasmid encoding the Na _V 1.7-CRS in an AAV virus. Treatment with this virus reduced Na _V 1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Transmembrane Ca _v 2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Ca _v 2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Ca _v 2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Ca _v 2.2–CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A ₁ R ₂ dipeptide in CBD3 as the anchoring Ca _v 2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization‐evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Ca _v 2.2 from CRMP2, reduced membrane Ca _v 2.2 expression and Ca ²⁺ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Ca _v 2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Vascular endothelial growth factor A (VEGF-A) is a pronociceptive factor that causes neuronal sensitization and pain. We reported that blocking the interaction between the membrane receptor neuropilin 1 (NRP1) and VEGF-A–blocked VEGF-A–mediated sensory neuron hyperexcitability and reduced mechanical hypersensitivity in a rodent chronic neuropathic pain model. These findings identified the NRP1-VEGF-A signaling axis for therapeutic targeting of chronic pain. In an in-silico screening of approximately 480 K small molecules binding to the extracellular b1b2 pocket of NRP1, we identified 9 chemical series, with 6 compounds disrupting VEGF-A binding to NRP1. The small molecule with greatest efficacy, 4′-methyl-2′-morpholino-2-(phenylamino)-[4,5′-bipyrimidin]-6(1H)-one, designated NRP1-4, was selected for further evaluation. In cultured primary sensory neurons, VEGF-A enhanced excitability and decreased firing threshold, which was blocked by NRP1-4. In addition, NaV1.7 and CaV2.2 currents and membrane expression were potentiated by treatment with VEGF-A, and this potentiation was blocked by NRP1-4 cotreatment. Neuropilin 1-4 reduced VEGF-A–mediated increases in the frequency and amplitude of spontaneous excitatory postsynaptic currents in dorsal horn of the spinal cord. Neuropilin 1-4 did not bind to more than 300 G-protein-coupled receptors and receptors including human opioids receptors, indicating a favorable safety profile. In rats with spared nerve injury–induced neuropathic pain, intrathecal administration of NRP1-4 significantly attenuated mechanical allodynia. Intravenous treatment with NRP1-4 reversed both mechanical allodynia and thermal hyperalgesia in rats with L5/L6 spinal nerve ligation–induced neuropathic pain. Collectively, our findings show that NRP1-4 is a first-in-class compound targeting the NRP1-VEGF-A signaling axis to control voltage-gated ion channel function, neuronal excitability, and synaptic activity that curb chronic pain.

Osteoarthritis (OA) is a highly prevalent and disabling joint disease, characterized by pathological progressive joint deformation and clinical symptoms of pain. Disease-modifying treatments remain unavailable, and pain-mitigation is often suboptimal, but recent studies suggest beneficial effects by inhibition of the voltage-gated sodium channel Na_V1.7. We previously identified compound 194 as an indirect inhibitor of Na_V1.7 by preventing SUMOylation of the Na_V1.7-trafficking protein, collapsin response mediator protein 2. Compound 194 reduces the functional activity of Na_V1.7 channels and produces effective analgesia in a variety of acute and neuropathic pain models. However, its effectiveness has not yet been evaluated in models of OA. Here, we explore the effects of 194 on pain-related outcomes in the OA-like monoiodoacetate model using behavioral assessment, biochemistry, novel in vivo fiber photometry, and patch clamp electrophysiology. We found that the monoiodoacetate model induced (1) increased pain-like behaviors and calcium responses of glutamatergic neurons in the parabrachial nucleus after evoked cold and mechanical stimuli, (2) conditioned place aversion to mechanical stimulation, (3) functional weight bearing asymmetry, (4) increased sodium currents in dorsal root ganglia neurons, and (5) increased calcitonin gene–related peptide-release in the spinal cord. Crucially, administration of 194 improved all these pain-related outcomes. Collectively, these findings support indirect inhibition of Na_V1.7 as an effective treatment of OA-related pain through the inhibition of collapsin response mediator protein 2-SUMOylation via compound 194.

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of intracellular GPCR signaling are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically encoded biosensors targeted to the plasma membrane and early endosomes were used to analyze compartmentalized signaling of protease-activated receptor 2 (PAR ₂ ); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to preferentially antagonize intracellular PAR ₂ signaling of pain. PAR ₂ agonists evoked sustained activation of PAR ₂ , Gαq, and β-arrestin-1 in early endosomes and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with targeted biosensors. Fluorescent dendrimer and core-shell polymeric NPs accumulated in endosomes of HEK293T cells, colonic epithelial cells, and nociceptors, detected by confocal microscopy. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR ₂ modulator. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR ₂ , Gαq, and β-arrestin-1 activation in early endosomes and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, fluorescent dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs accumulated in neurons, sites of PAR ₂ expression. Both NP formulations of AZ3451, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. These results provide evidence that PAR ₂ endosomal signaling mediates pain and that nanomedicines that antagonize PAR ₂ in endosomes effectively relieve pain. NP-mediated delivery may improve the efficacy of other GPCR antagonists for treatment of diverse diseases.