C2230, a preferential use- and state-dependent CaV2.2 channel blocker, mitigates pain behaviors across multiple pain models
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
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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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 13

Locations of alanine-scanning mutation sites on CaV 2.2.

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Locations of alanine-scanning mutation sites on CaV 2.2. (A) Surface rep...
(A) Surface representation of the human CaV2.2 α subunit (PDB: 7VFV(40)) with bound PD173212 in stick representation. View facing the open D-III/D-IV fenestration. (B) Ribbon representation through the D-III/D-IV fenestration with the alanine mutation sites on the D-III S5, S6 and D-IV S6 helices shown as spheres. The mutations affecting inhibition by C2230 are shown as sticks and labeled according to their rat CaV2.2 sequence, with the corresponding human numbering in parentheses. Residues important for PD173212 binding (40) also shown as sticks and their labels are underlined. (C) Close up views showing all amino acids with their rat sequence numbers. (D–F) Bar graphs of percent inhibition by 20 μM C2230 for alanine scan of D-III S5 (D), D-IV S6 (E), and D-III S6 (F) helices. Data in red bars indicate mutations affecting inhibition by C2230 versus WT while gray bars denote mutations that were not different from WT. Mutations of L1288A, A1294G, S1390A, F1404A, and F1683A significantly reduced inhibition C2230 when compared with the WT. Channels were clamped at –80 mV and currents were elicited by depolarization to + 10 mV. P values are as indicated, Kruskal-Wallis test followed by Dunn’s post hoc test; n = 4–12 cells from 2–3 independent experiments. (G) The fold change in IC50 values for C2230 inhibiting the specified mutants is presented relative to its inhibition of the WT CaV 2.2 channel, assessed at holding potentials of –80 mV and –50 mV. Note the IC50s of C2230 on these mutants at –80 mV holding potential were calculated using the Hill equation (IC50 = [C2230] × Ires/(1 – Ires)), where Ires for each mutant equals to ‘1 – inhibition ratio’ as determined in D–F. The IC50s of C2230 on these mutants at –50 mV holding, however, were experimentally determined (n = 5–6). See Supplemental Table 3 for full statistical analysis.