Protein-protein interaction inhibitor of the sodium NaV1.7 channel has analgesic effects in preclinical models of pain.

Voltage-gated T-type Ca²⁺(Ca_V3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates Ca_V3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant Ca_V3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate Ca_V3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate Ca_V3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENTNeuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca²⁺channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of Ca_V3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

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.

The sodium channel Na_v1.7 is a master regulator of nociceptive input into the central nervous system. Mutations in this channel can result in painful conditions and produce insensitivity to pain. Despite being recognized as a “poster child” for nociceptive signaling and human pain, targeting Na_v1.7 has not yet produced a clinical drug. Recent work has illuminated the Na_v1.7 interactome, offering insights into the regulation of these channels and identifying potentially new druggable targets. Among the regulators of Na_v1.7 is the cytosolic collapsin response mediator protein 2 (CRMP2). CRMP2, modified at lysine 374 (K374) by addition of a small ubiquitin-like modifier (SUMO), bound Na_v1.7 to regulate its membrane localization and function. Corollary to this, preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in rats with neuropathic pain. Notably, loss of CRMP2 SUMOylation did not compromise other innate functions of CRMP2. To further elucidate the in vivo role of CRMP2 SUMOylation in pain, we generated CRMP2 K374A knock-in (CRMP2^K374A/K374A) mice in which Lys374 was replaced with Ala. CRMP2^K374A/K374A mice had reduced Na_v1.7 membrane localization and function in female, but not male, sensory neurons. Behavioral appraisal of CRMP2^K374A/K374A mice demonstrated no changes in depressive or repetitive, compulsive-like behaviors and a decrease in noxious thermal sensitivity. No changes were observed in CRMP2^K374A/K374A mice to inflammatory, acute, or visceral pain. By contrast, in a neuropathic model, CRMP2^K374A/K374A mice failed to develop persistent mechanical allodynia. Our study suggests that CRMP2 SUMOylation–dependent control of peripheral Na_v1.7 is a hallmark of chronic, but not physiological, 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.

Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltage-gated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.