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.

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.

The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA_A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α₆-containing GABA_A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α₆-containing GABA_A receptor blockade or transient α₆-containing GABA_A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α₆ subunit is expressed in IB4⁺ and CGRP⁺ primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α₆ subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α₆-containing GABA_A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α₆ subunit reduces tactile allodynia and restores α₆ subunit expression in neuropathic rats. Positive allosteric modulators of the α₆-containing GABA_A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α₆ subunit is expressed in humans. This receptor is found in CGRP⁺ and P2X3⁺ primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α₆-containing GABA_A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.