C2230, a preferential use- and state-dependent CaV2.2 channel blocker, mitigates pain behaviors across multiple pain models
Cheng Tang, … , Olga A. Korczeniewska, Rajesh Khanna
Cheng Tang, … , Olga A. Korczeniewska, Rajesh Khanna
Published December 10, 2024
Citation Information: J Clin Invest. 2025;135(4):e177429. https://doi.org/10.1172/JCI177429.
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Research Article Neuroscience

C2230, a preferential use- and state-dependent CaV2.2 channel blocker, mitigates pain behaviors across multiple pain models

Abstract

Antagonists — such as Ziconotide and Gabapentin — of the CaV2.2 (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 CaV2.2. Here, we report the identification of C2230, an aryloxy-hydroxypropylamine, as a CaV2.2 blocker. C2230 trapped and stabilized inactivated CaV2.2 in a slow-recovering state and accelerated the open-state inactivation of the channel, conferring an advantageous use-dependent inhibition profile. C2230 inhibited CaV2.2 during high-frequency stimulation, while sparing other voltage-gated ion channels. C2230 inhibited CaV2.2 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 CaV2.2 blockers, making it a promising lead compound for analgesic development.

Authors

Cheng Tang, Kimberly Gomez, Yan Chen, Heather N. Allen, Sara Hestehave, Erick J. Rodríguez-Palma, Santiago Loya-Lopez, Aida Calderon-Rivera, Paz Duran, Tyler S. Nelson, Siva Rama Raju Kanumuri, Bijal Shah, Nihar R. Panigrahi, Samantha Perez-Miller, Morgan K. Schackmuth, Shivani Ruparel, Amol Patwardhan, Theodore J. Price, Paramjit S. Arora, Ravindra K. Sharma, Abhisheak Sharma, Jie Yu, Olga A. Korczeniewska, Rajesh Khanna

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Figure 2

Use- and state-dependent inhibition of CaV2.2 by C2230.

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Use- and state-dependent inhibition of CaV2.2 by C2230. (A) Voltage prot...
(A) Voltage protocols assessing activation (P1), steady-state inactivation (P2), the development of time-dependent inactivation (P3) of CaV2.2 channels. (B) CaV2.2 current-voltage relationships before (DMSO 0.1%) and after C2230 (10 μM) treatment. Currents in each recording cell were normalized to the maximum peak current before C2230 treatment (blue and orange solid curves) or its own maximum peak current (orange dashed curve) (n = 13 cells). (C) Steady-state activation and inactivation relationships of CaV2.2 channels in the absence and presence of 10 μM C2230 (n = 12 cells). (D) Time-dependent development of CaV2.2 channels’ closed-state inactivation in the absence and presence of 20 μM C2230 (n = 12–13 cells, P values as indicated, Unpaired t test). (E and F) Mean normalized current traces (left) and bar graphs (right) of τ of inactivation (s) at Vh of –80 mV (E) and –50 mV (F) (n = 8–10 cells, P values as indicated, Paired t test). (G) Time-dependent current decay of CaV2.2 channels during 60 consecutive step depolarizations at frequencies of 1, 3, and 10 Hz, with and without 10 μM C2230. The upper panel depicts the typical current traces at the first and the 60th depolarization in each group (n = 9–11 cells) while the summary data is shown in the lower panels. (H) Time-dependent recovery of CaV2.2 channels from inactivated state as evaluated using the voltage protocol (upper panel; n = 14 cells) with the time constants for fast recovery (τfast) and slow recovery (τslow) being increased from 0.151 ± 0.022 s to 0.232 ± 0.026 s, and 5.719 ± 1.079 s to 9.382 ± 1.079 s by C2230 treatment, respectively (P < 0.05 for both τfast and τslow comparisons, Mann-Whitney test). The proportion of fast recovering channels were reduced in the C2230 group compared with that in the DMSO group (66.7 ± 3.0% to 42.4 ± 2.0%; P < 0.001; Mann-Whitney test). All data are from at least 3 independent experiments. See Supplemental Table 3 for full statistical analysis.