Targeting the CaVα-CaVβ interaction yields an antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy

Abstract

Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, β, γ and α2δ subunits. The β subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVβ's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the β subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVβ and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVβ function and ultimately be developed as a nonopioid therapeutic for chronic pain.

Figure 1.

Structure-guided high-throughput screen of CaVα–CaVβ interaction inhibitors identifies IPPQ (A) Space-filling model of the CaVβ2a subunit showing its α-binding pocket (ABP). A ribbon representation of the CaVα-interaction domain (AID, purple) and the AID’s key interaction residue (M245) are highlighted. (B) Ball-and-stick representation of key ABP residues is shown (ie, V241, I343, and N390). (C) A 3-dimensional representation of IPPQ (red) overlaid on the AIDs reveals shared chemical features, especially at AID residues W440, I441, and Y437. (D) 1D ¹H STD NMR showing on-resonance difference spectrum for IPPQ (red); only regions that yielded a STD signal in the presence of IPPQ are plotted. The 2-dimensional chemical structure of compound IPPQ shows that region a corresponds with the STD signal highlighted. (E) Amino acid alignments of AID residues of the indicated calcium channel subtypes. Residues different from the CaV2.2 core AID sequence are denoted in orange. Asterisks denote the key residues conserved in all AID sequences involved in the interaction with the beta subunits. Microscale thermophoresis was used to determine dissociation constants for binding of AID-CaV2.2 (F), CaV2.1 (G), CaV1.1 (H), CaV1.2/3/4 (I) (500–0.01 nM), and CaV2.3 (J) to Beta2-CaVβ2-His (25 nM) in the presence (filled red circles) or absence of 10-μM IPPQ (open squares). Data are presented as mean ± SEM. The data were all fit with a one-site binding model, and the K_d and r² values are indicated. The data for Beta2-CaVβ2-His and AID-CaV2.1 in the presence of vehicle also fit well with a 2-site binding model (r² = 0.84) but yielded K_ds that were a 1000-fold higher than the one-binding site equation. In the presence of IPPQ, the data in panel (G) had a better fit (r² = 0.74) but yielded K_ds values of 2.3 nM (for the high affinity site) and ~81 mM (for the low-affinity site). (K) Concentration–response curve illustrates that IPPQ inhibits membrane depolarization-evoked Ca²⁺ influx in DRGs (n > 150 cells per concentration from 3 independent platings) in a concentration-dependent manner. IPPQ was applied overnight (15–18 hours). AID, alpha interaction domain; CaV, voltage-gated calcium.

Figure 2.

Constellation pharmacology to identify potential off-target effects of IPPQ. Ca²⁺ imaging from DRGs treated overnight (15–18 hours) with 0.1% DMSO (n = 1152) or 20-μM compound IPPQ (n = 1359). These data include DRGs analyzed over 2 independent experiments with up to 9 trials per experiment. During each trial, >100 DRGs were sequentially stimulated for ~15 seconds (indicated by colored bars) with menthol (400 nM), histamine (50 μM), ATP (10 μM), allyl isothiocyanate (AITC) (200 μM), acetylcholine (1 mM), capsaicin (100 nM), and KCl (90 mM). Only DRGs responsive to membrane depolarization-evoked Ca²⁺ influx by KCl were included. Furthermore, only responses greater than 10% of baseline were included (indicated by filled black circles). (A) Analysis of DRG responses revealed the percentage of cells responding to the indicated number of molecular agonists, independent of which agonists elicited a response, in both DMSO- and IPPQ-treated conditions (n = 184–588, *P < 0.001, z-test.) (B) Analysis of DRG responses revealed the percentage of cells responding to the indicated molecular agonist, independent of any other agonist that also elicited a response, in both DMSO- and IPPQ-treated DRGs (n = 88–693, *P < 0.01, z-test). (C) Bar graph of average peak response to each of the indicated trigger. (D) Bar graph of the average peak response to KCl in DRG neurons responsive to the indicated trigger (n = 1152–1359, *P < 0.05, Student t test vs 0.1% DMSO). DRG, dorsal root ganglion.

Figure 3.

IPPQ disrupts α1B–β1b, α1B–β2a, and α1B–β3–α2δ−1 interactions to inhibit Ca²⁺ currents. HEK293 cells expressing either (A) α1B + β1b, (B) α1B + β2a, (C) α1B + β2a + α2δ−1, (D) α1B + β3 + α2δ−1, or (E) α1B + β4 + α2δ−1 were subjected to an activation voltage step protocol to determine their I-V relationship after overnight (15–18 hours) treatment with 0.1% DMSO (black) or 20-μM IPPQ (red). Ca²⁺ currents in α1B + β1b, α1B + β2a, α1B + β2a + α2δ−1, and α1B + β3 + α2δ−1 expressing cells were significantly reduced in IPPQ-treated DRGs compared with 0.1% DMSO-treated counterparts (n = 5–18, *P < 0.05, Kruskal–Wallis test with Dunnett post hoc comparisons).

Figure 4.

IPPQ preferentially inhibits Ca²⁺ currents mediated by CaV2.2. (A) Activation (ie, current–voltage [I-V] relationship voltage step protocol). Cells were held at resting membrane potential for 5-ms before depolarization by 200-ms voltage steps from −70 to +60 mV in 10-mV increments. Currents were normalized to each cell’s capacitance (pF). This allowed for collection of current density data to analyze activation of Ca²⁺ channels as a function of current vs voltage and peak current density. (B) Representative Ca²⁺ current traces from DRGs subjected to the activation protocol (shown in A). (C) Summary graph of peak Ca²⁺ current density (pA/pF) from DRGs incubated with 0.1% DMSO or 20-μM IPPQ overnight (15–18 hours) (n = 12; *P < 0.05, Kruskal–Wallis test with Dunnett post hoc comparisons). (D) Inactivation voltage-step protocol. Cells were held at −90 mV for 20 ms before depolarization by 1.5-second voltage steps from −100 to +10 mV in 10-mV increments, followed by a 20-ms pulse at 10 mV before returning to −90 mV for 20 ms. (E) Normalized peak current plotted against its preceding holding potential and fitted with the Boltzmann relation. No significant differences were detected in half-maximal voltage or slope properties of either activation or inactivation between 0.1% DMSO and IPPQ conditions. (F) Summary graph of peak Ca²⁺ current density (pA/pF). Presence of IPPQ did not significantly reduce Ca²⁺ currents pharmacologically isolated through L-type, P/Q-type, or R-type Ca²⁺ channels. Presence of IPPQ significantly reduced N-type Ca²⁺ currents (compared with DMSO-treated DRGs, n = 6–10; *P < 0.05, Kruskal–Wallis test with Dunnett post hoc comparisons).

Figure 5.

IPPQ does not bind to opioid receptors and does not affect G protein–mediated inhibition of Ca²⁺ current in rat sensory neurons. Competition radioligand binding was performed in CHO cells expressing the human opioids receptors MOR (A), DOR (B), or KOR (C). Compound IPPQ or a positive control compound competed against ³H-diprenorphine in all 3 cell lines. Curves reported as the mean ± SEM of the mean value from each individual experiment in N = 3 independent experiments. The K_I also reported as the mean ± SEM of the individual value from each of N = 3 independent experiments. (A) Compound IPPQ did not bind to the MOR. Naloxone K_I = 33.9 ± 1.7 nM. (B) Compound IPPQ produced 76.2% inhibition at 10 μM at the DOR, with an incomplete curve. This indicates an IC₅₀ >3.3 μM and a K_I >1 μM. Naloxone K_I = 55.7 ± 6.7 nM. (C) Compound IPPQ did not appreciably bind to the KOR. U50488 K_I = 22.4 ± 4.1 nM. (D) Representative current traces following the “double pulse” protocol (top) for control, IPPQ (20 μM) applied for 30 minutes, and norepinephrine (NE, 10 μM). (E) Histograms of ratio of I₂/I_1, measured at 10 msec after the step to −10 mV of DRG neurons treated with 0.1% DMSO, IPPQ, or NE. *P, 0.05, Kruskal–Wallis test with Dunnett post hoc comparisons. DRG, dorsal root ganglion.

Figure 6.

IPPQ decreases spinal excitatory synaptic transmission. Photomicrograph of slice preparation showing that the substantia gelatinosa (SG) can be identified as a translucent pale band in the superficial dorsal horn enabling positioning of the recording electrode to this region. (A, i) infrared differential interference contrast image (IR-DIC) and (A, ii) image of the same cell (indicated by a purple red box in middle panel) with part of the recording electrode after whole-cell configuration. (B) Representative recording of spontaneous and miniature EPSCs traces of cells from the indicated groups: 0.1% DMSO or 20-μM IPPQ, applied through perfusion system during the experiment within 3 hours. The recordings were obtained within 30 minutes to 3 hours of drug perfusion; no difference was observed between recordings performed earlier vs later in the window after drug perfusion (data not shown). (C and D) The cumulative probability and summary of sEPSC amplitudes. (E and F) The cumulative probability of interevent interval and frequencies of sEPSCs for the indicated groups are shown. IPPQ treatment had no effect on the amplitude but decreased the frequency of the recorded sEPSCs. (G and H) The cumulative probability and summary of mEPSC amplitudes. (I and J) The cumulative probability of interevent interval and frequencies of mEPSCs. Both the frequency and amplitude of mEPSCs were decreased by 20-μM IPPQ. Data are expressed as mean ± SEM from n = 16 to 18 cells per condition. *P < 0.05 (vs DMSO); Student t test.

Figure 7.

IPPQ increases paired-pulse ratios. (A) Representative traces of paired-pulse ratio (PPR) from the indicated groups: 0.1% DMSO or 20-μM IPPQ. The recordings were obtained within 30 minutes to 3 hours of drug perfusion. (B) Summary of average PPRs from the indicated groups. Data are expressed as mean ± SEM from n = 13 to 17 cells per condition. *P < 0.05 (vs DMSO); Student t test.

Figure 8.

IPPQ decreases CaV2.2 presynaptic localization in vivo and reduces evoked CGRP release from spinal cord. (A) Immunoblots showing the integrity of the synaptic fractionation from lumbar dorsal horn of the spinal cord. The non-postsynaptic density (non-PSD) fraction was enriched in the presynaptic marker synaptophysin (Syp), and the PSD fraction was enriched in the postsynaptic marker PSD95. The membrane-associated protein flotillin was used as a loading control. (B) Immunoblots showing the presynaptic CaV2.2 levels in the lumbar dorsal horn of the spinal cord of animals having received IPPQ (2 μg in 5 μL, intrathecally) compared with vehicle (0.1% DMSO). The synaptosomes were prepared from the dorsal horn of the spinal cord of rats 1 hour after injection with IPPQ. Synaptophysin shows the integrity of each fraction. Flotillin is used as a loading control. (C) Bar graph showing decreased CaV2.2 levels at the presynaptic sites of lumbar dorsal horn of the spinal cord in IPPQ-treated animals. Mean ± SEM, *P < 0.05, Mann–Whitney compared with the DMSO vehicle treatment.

Figure 9.

IPPQ acutely reduces calcium influx and evoked calcitonin gene–related peptide (CGRP) release from spinal cord. (A) Representative traces of membrane depolarization-evoked Ca²⁺ influx by 90-mM KCl before and after 10 minutes application of either 0.1% DMSO (A) or 20-μM IPPQ (B). (C) Quantification of the peak ratio for the indicated condition. Ten-minute treatment with IPPQ significantly reduced the peak ratio (vs 0.1% DMSO, n = 460–493 cells, *P < 0.05, Student t-test). (D) CGRP released from spinal cord fractions collected every 10 minutes corresponded with the following conditions: 1-baseline, 2-baseline, 3-treatment, 4-treatment + 90-mM KCl, 5 wash. Previous treatment for 10 minutes with compound IPPQ significantly reduced high potassium-evoked CGRP release in spinal cord (vs DMSO, n = 4–5, *P < 0.0001, one-way ANOVA with Dunnett post hoc comparisons). ANOVA, analysis of variance.

Figure 10.

IPPQ is antinociceptive in naive animals and reverses mechanical allodynia and thermal hyperalgesia in acute and neuropathic pain models. (A) Paw withdrawal latencies (PWLs) of naive rodents to a heat stimulus were significantly increased 1 hour after an intrathecal (i.t.) injection of IPPQ (2 μg) compared to animals injected with vehicle (10% DMSO, 10% Tween-80, 80% saline) (compared with i.t. injection of vehicle (n = 6, *P < 0.05, 2-way ANOVA). (B) PWLs of rodents subjected to targeted Cas9-mediated editing of Nf1 (using i.t. injection) were significantly decreased, but i.t. injections of IPPQ reversed this behavior (vs i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (C) PWLs of rodents that received a paw incision (Sx) on the left hind paw were significantly decreased. Intrathecal injection of IPPQ reversed this nociceptive behavior (compared with i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (D) Paw withdrawal thresholds (PWTs) of rodents that received a paw incision (Sx) on the left hind paw were significantly decreased. Intrathecal injection of IPPQ reversed this nociceptive behavior (compared with i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (E) PWLs of rodents that received a spinal nerve ligation (SNL) on the left hind paw were significantly decreased. Intrathecal injection of IPPQ reversed this nociceptive behavior (compared with i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (F) PWTs of SNL rodents were also significantly decreased. Intrathecal injection of IPPQ also reversed this behavior (vs i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (G) PWTs of rodents that received four 2-mg/kg intraperitoneal (i.p.) injections of paclitaxel (Px) over 12 days were significantly decreased. Intrathecal injection of IPPQ reversed this behavior (vs i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). (H) PWTs of rodents were significantly decreased 15 days after receiving 3 i.t. injections of gp-120. Intrathecal injection of IPPQ reversed this behavior (vs i.t. injection of vehicle, n = 6, *P < 0.05, 2-way ANOVA). The experiments were conducted in a blinded fashion. ANOVA, analysis of variance.

Figure 11.

IPPQ is analgesic in mice but does not affect anxiety-related behaviors. (A) Paw withdrawal latencies of naive mice in a hot-plate test (52°C) 75 minutes after injection of IPPQ (15 mg/kg) administered intraperitoneally (i.p.) were unaffected (compared with i.p. injection of vehicle: 10% DMSO, 10% Tween-80, 80% saline), n = 12, P = 0.7583, t test with the Welch correction. (B) Tail-flick latencies of naive mice to a hot (52°C) water bath 75 minutes after injection of IPPQ administered i.p. were significantly increased (vs vehicle i.p. injection), n = 12, *P < 0.05, t test with the Welch correction). (C) Paw withdrawal latencies of naive mice to the hot-plate test (52°C) 60 minutes after i.p. injection of gabapentin (100 mg/kg) administered were increased (compared with i.p. injection of vehicle), n = 9, *P < 0.05, Mann–Whitney test. (D) Tail-flick latencies of naive mice to a hot (52°C) water bath 60 minutes after i.p. injection of gabapentin (100 mg/kg) administered were unaffected (vs vehicle i.p. injection), n = 9, P = 0.445, t test with the Welch correction). (E) Naive mice received an i.p. injection of IPPQ (15 mg/kg), or its vehicle and anxiety-related behaviors were assessed during 10 minutes in an elevated plus maze test. Heatmaps of the positions occupied in the elevated plus maze apparatus by treatment groups are represented in (E). (F) Bar graphs of the mean time spent in the open arms of the elevated plus maze. No differences were found between animals treated with IPPQ or with its vehicle (P = 0.6014, t test with the Welch correction, n = 12 mice/group). All experiments were conducted in a blinded fashion.