Antagonists — such as Ziconotide and Gabapentin — of the Ca _V 2.2 (N-type) calcium channels are used clinically as analgesics for chronic pain. However, their use is limited by narrow therapeutic windows, difficult dosing routes (Ziconotide), misuse, and overdoses (Gabapentin), as well as a litany of adverse effects. Expansion of novel pain therapeutics may emerge from mechanism-based interrogation of Ca _V 2.2. Here, we report the identification of C2230, an aryloxy-hydroxypropylamine, as a Ca _V 2.2 blocker. C2230 trapped and stabilized inactivated Ca _V 2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited Ca _V 2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited Ca _V 2.2 in dorsal root and trigeminal ganglia neurons from rats, marmosets, and humans in a G-protein-coupled-receptor–independent manner. Further, C2230 reduced evoked excitatory postsynaptic currents and excitatory neurotransmitter release in the spinal cord, leading to relief of neuropathic, orofacial, and osteoarthritic pain-like behaviors via 3 different routes of administration. C2230 also decreased fiber photometry-based calcium responses in the parabrachial nucleus, mitigated aversive behavioral responses to mechanical stimuli after neuropathic injury, and preserved protective pain responses, all without affecting motor or cardiovascular function. Finally, site-directed mutation analysis demonstrated that C2230 binds differently than other known Ca _V 2.2 blockers, making it a promising lead compound for analgesic development.

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.

Oral cancer causes pain associated with cancer progression. We report here that the function of the Ca ²⁺ channel ORAI1 is an important regulator of oral cancer pain. ORAI1 was highly expressed in tumor samples from patients with oral cancer, and ORAI1 activation caused sustained Ca ²⁺ influx in human oral cancer cells. RNA-seq analysis showed that ORAI1 regulated many genes encoding oral cancer markers such as metalloproteases (MMPs) and pain modulators. Compared with control cells, oral cancer cells lacking ORAI1 formed smaller tumors that elicited decreased allodynia when inoculated into mouse paws. Exposure of trigeminal ganglia neurons to MMP1 evoked an increase in action potentials. These data demonstrate an important role of ORAI1 in oral cancer progression and pain, potentially by controlling MMP1 abundance.

Dysregulation of voltage-gated sodium Na_V1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/Na_V1.7 interaction and increased functional activity of Na_V1.7. Targeting this feed-forward regulation, we developed compound 194, which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of Na_V1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we used a comprehensive array of approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of Na_V1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a, Dpysl2, and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and Na_V1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/Na_V1.7 interaction, impeded Na_V1.7 diffusion on the plasma membrane, and subsequently diminished Na_V1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve, 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating Na_V1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.