Seven novel modulators of the analgesic target NaV 1.7 uncovered using a high-throughput venom-based discovery approach

Background and Purpose

Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel Na_V1.7 (hNa_V1.7) are indifferent to pain, making hNa_V1.7 a promising target for analgesic development. Since spider venoms are replete with Na_V channel modulators, we examined their potential as a source of hNa_V1.7 inhibitors.

Experimental Approach

We developed a high-throughput fluorescent-based assay to screen spider venoms against hNa_V1.7 and isolate ‘hit’ peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNa_V1.7 was transplanted into the homotetrameric K_V2.1 channel.

Key Results

We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNa_V1.7. By deconvoluting ‘hit’ venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNa_V1.7 with a high level of selectivity over all other subtypes, except hNa_V1.1. We showed that Hd1a is a gating modifier that inhibits hNa_V1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability.

Conclusion and Implications

Our data indicate that spider venoms are a rich natural source of hNa_V1.7 inhibitors that might be useful leads for the development of novel analgesics.     

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

Isolation,synthesis and characterization of ω-TRTX-Cc1a,a novel tarantula venom peptide that selectively targets L-type CaV channels

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (Ca_V) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of Ca_V2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba²⁺ currents (I_Ba) mediated by L-type (Ca_V1.2 and Ca_V1.3) Ca_V channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC₅₀) values of 825 nM and 2.24 μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I_Ba mediated by high voltage-activated Ca_V channels but did not affect low voltage-activated T-type Ca_V channels. Cc1a exhibited weak activity at Na_V1.5 and Na_V1.7 voltage-gated sodium (Na_V) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1–E5) or C-terminus (ΔW35–V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type Ca_V channels.     

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors

BACKGROUND AND PURPOSE

The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ-conopeptide (µ-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant.

EXPERIMENTAL APPROACH

µ-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data.

KEY RESULTS

Synthetic µ-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC₅₀= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na_V1.4 (IC₅₀= 1.3 nM) and Na_V1.2 channels in a long-lasting manner. Cardiac Na_V1.5 and DRG-specific Na_V1.8 channels were not blocked at 1 µM. µ-CnIIIC also blocked the α3β2 nAChR subtype (IC₅₀= 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to α-conotoxins acting on nAChRs.

CONCLUSION AND IMPLICATIONS

µ-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.     

Fusion of Ssm6a with a protein scaffold retains selectivity on NaV1.7 and improves its therapeutic potential against chronic pain

Voltage‐gated sodium channel Na_V1.7 serves as an attractive target for chronic pain treatment. Several venom peptides were found to selectively inhibit Na_V1.7 but with intrinsic problems. Among them, Ssm6a, a recently discovered centipede venom peptide, shows the greatest selectivity against Na_V1.7, but dissociates from the target too fast and loses bioactivity in synthetic forms. As a disulfide‐rich venom peptide, it is difficult to optimize Ssm6a by artificial mutagenesis and produce the peptide with common industrial manufacturing methods. Here, we developed a novel protein scaffold fusion strategy to address these concerns. Instead of directly mutating Ssm6a, we genetically fused Ssm6a with a protein scaffold engineered from human muscle fatty acid‐binding protein. The resultant fusion protein, SP‐TOX, maintained the selectivity and potency of Ssm6a upon Na_V1.7 but dissociated from target at least 10 times more slowly. SP‐TOX dramatically reduced inflammatory pain in a rat model through DRG‐targeted delivery. Importantly, SP‐TOX can be expressed cytosolically in Escherichia coli and purified in a cost‐effective way. In summary, our study provided the first example of cytosolically expressed fusion protein with high potency and selectivity on Na_V1.7. Our protein scaffold fusion approach may have its broad application in optimizing disulfide‐rich venom peptides for therapeutic usage.     

Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a) Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

Voltage-gated sodium channels (VGSCs; Na_V1.1–Na_V1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC₅₀ values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on Na_V1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC₅₀ value of 5.12 nM, compared with IC₅₀ values of 61.7–2700 nM for other heterologously expressed Na_V1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on Na_V1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.     

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K_Vs), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus laevis oocytes: 13 cloned voltage-gated potassium channels (K_V1.1–K_V1.6, K_V1.1 triple mutant, K_V2.1, K_V3.1, K_V4.2, K_V4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na_V1.2–Na_V1.8 and the insect channel DmNa_V1). Our data show that APEKTx1 selectively blocks K_V1.1 channels in a very potent manner with an IC₅₀ value of 0.9 nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124 nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K_V1.1 channels with that of a competitive inhibitor of trypsin.     

Isolation and characterization of two novel scorpion toxins: The α-toxin-like CeII8, specific for Nav1.7 channels and the classical anti-mammalian CeII9, specific for Nav1.4 channels

Scorpion β-toxins represent a particular pharmacological group of voltage-gated sodium channel (VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. Here, we report the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Thorell, 1876), which is responsible for deadly cases of intoxication in Mexico. The soluble venom was fractionated by gel filtration and ion-exchange chromatography, followed by reversed-phase HPLC. The toxins CeII8 and CeII9 were further purified and both their amino acid sequence and molecular weight were determined. Both toxins were electrophysiologically characterized on four mammalian VGSCs (rNa_v1.2, rNa_v1.4, hNa_v1.5 and rNa_v1.7) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Although CeII8 has the highest sequence similarity with scorpion α-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear β-toxin effect and was selective towards Na_v1.7, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first β-toxin active on Na_v1.7. CeII9, a typical anti-mammalian β-toxin, selectively modulated Na_v1.4 at a concentration of 700 nM and was, in contrast to CeII8, found to be lethal to mice. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the β-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.     

On the relationship between block of the cardiac Na⁺ channel and drug-induced prolongation of the QRS complex

BACKGROUND AND PURPOSE

Inhibition of the human cardiac Na⁺ channel (hNa_v1.5) can prolong the QRS complex and has been associated with increased mortality in patients with underlying cardiovascular disease. The safety implications of blocking hNa_v1.5 channels suggest the need to test for this activity early in drug discovery in order to design out any potential liability. However, interpretation of hNa_v1.5 blocking potency requires knowledge of how hNa_v1.5 block translates into prolongation of the QRS complex.

EXPERIMENTAL APPROACH

We tested Class I anti-arrhythmics, other known QRS prolonging drugs and drugs not reported to prolong the QRS complex. Their block of hNa_v1.5 channels (as IC₅₀ values) was measured in an automated electrophysiology-based assay. These IC₅₀ values were compared with published reports of the corresponding unbound (free) plasma concentrations attained during clinical use (fC_max) to provide an IC₅₀ : fC_max ratio.

KEY RESULTS

For 42 Class I anti-arrhythmics and other QRS prolonging drugs, 67% had IC₅₀ : fC_max ratios <30. For 55 non-QRS prolonging drugs tested, 72% had ratios >100. Finally, we determined the relationship between the IC₅₀ value and the free drug concentration associated with prolongation of the QRS complex in humans. For 37 drugs, QRS complex prolongation was observed at free plasma concentrations that were about 15-fold lower than the corresponding IC₅₀ at hNa_v1.5 channels.

CONCLUSIONS AND IMPLICATIONS

A margin of 30- to 100-fold between hNa_v1.5 IC₅₀ and fC_max appears to confer an acceptable degree of safety from QRS prolongation. QRS prolongation occurs on average at free plasma levels 15-fold below the IC₅₀ at hNa_v1.5 channels.

LINKED ARTICLE

This article is commented on by Gintant et al., pp. 254–259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01433.x     

Characterization of a Novel BmαTX47 Toxin Modulating Sodium Channels: The Crucial Role of Expression Vectors in Toxin Pharmacological Activity

Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin BmαTX47 from the scorpion Buthus martensii Karsch. The recombinant BmαTX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological experiments showed that the recombinant BmαTX47 was a new α-scorpion toxin which could inhibit the fast inactivation of rNa_v1.2, mNa_v1.4 and hNa_v1.5 channels. Importantly, the different expression vectors were found to strongly affect BmαTX47 pharmacological activities while toxins were obtained by the same expression and purification procedures. When 10 µM recombinant BmαTX47 from the pET-28a vector was applied, the values of I_5ms/I_peak for rNa_v1.2, mNa_v1.4 and hNa_v1.5 channels were 44.12% ± 3.17%, 25.40% ± 4.89% and 65.34% ± 3.86%, respectively, which were better than those values of 11.33% ± 1.46%, 15.96% ± 1.87% and 5.24% ± 2.38% for rNa_v1.2, mNa_v1.4 and hNa_v1.5 channels delayed by 10 µM recombinant BmαTX47 from the pET-14b vector. The dose-response experiments further indicated the EC₅₀ values of recombinant BmαTX47 from the pET-28a vector were 7262.9 ± 755.9 nM for rNa_v1.2 channel and 1005.8 ± 118.6 nM for hNav1.5 channel, respectively. Together, these findings highlighted the important role of expression vectors in scorpion toxin pharmacological properties, which would accelerate the understanding of the structure-function relationships of scorpion toxins and promote the potential application of toxins in the near future.     

Co-expression of NaVβ subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins

Background and Purpose

Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (Na_V1) and one or two accessory β-subunits (Na_Vβs). Neurons in mammals can express one or more of seven isoforms of Na_V1 and one or more of four isoforms of Na_Vβ. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in Na_V1. Hitherto, the effects of Na_Vβ-subunit co-expression on the activity of these toxins have not been comprehensively assessed.

Experimental Approach

Four μ-conotoxins (μ-TIIIA, μ-PIIIA, μ-SmIIIA and μ-KIIIA), TTX and STX were tested against Na_V1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of Na_Vβ1, β2, β3 or β4 and, for Na_V1.7, binary combinations of thereof.

Key Results

Co-expression of Na_Vβ-subunits modifies the block by μ-conotoxins: in general, Na_Vβ1 or β3 co-expression tended to increase k_on (in the most extreme instance by ninefold), whereas Na_Vβ2 or β4 co-expression decreased k_on (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by Na_Vβ-subunit co-expression. Tests of Na_Vβ1 : β2 chimeras co-expressed with Na_V1.7 suggest that the extracellular portion of the Na_Vβ subunit is largely responsible for altering μ-conotoxin kinetics.

Conclusions and Implications

These results are the first indication that Na_Vβ subunit co-expression can markedly influence μ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. μ-Conotoxins could, in principle, be used to pharmacologically probe the Na_Vβ subunit composition of endogenously expressed VGSCs.     

The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000–4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na⁺, K⁺ and Ca²⁺ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na⁺ currents and T-type Ca²⁺ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD₅₀ was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.     

Alkaloids from Veratrum taliense Exert Cardiovascular Toxic Effects via Cardiac Sodium Channel Subtype 1.5

Several species of the genus Veratrum that produce steroid alkaloids are commonly used to treat pain and hypertension in China and Europe. However, Veratrum alkaloids (VAs) induce serious cardiovascular toxicity. In China, Veratrum treatment often leads to many side effects and even causes the death of patients, but the pathophysiological mechanisms under these adverse effects are not clear. Here, two solanidine-type VAs (isorubijervine and rubijervine) isolated from Veratrum taliense exhibited strong cardiovascular toxicity. A pathophysiological study indicated that these VAs blocked sodium channels Na_V1.3–1.5 and exhibited the strongest ability to inhibit Na_V1.5, which is specifically expressed in cardiac tissue and plays an essential role in cardiac physiological function. This result reveals that VAs exert their cardiovascular toxicity via the Na_V1.5 channel. The effects of VAs on Na_V1.3 and Na_V1.4 may be related to their analgesic effect and skeletal muscle toxicity, respectively.     

Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade

Introduction

The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa_V1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture.

Methods

Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds.

Results

Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC₅₀s (paired values denote constantly cultured and frozen, respectively) of 4.8 ± 0.4 and 5.1 ± 0.4, 1.4 ± 0.1 and 1.1 ± 0.1, 24.4 ± 2.4 and 21.9 ± 1.8, 2.1 ± 0.4 and 2.1 ± 0.1, 5.2 ± 0.4 and 4.0 ± 0.2 μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa_V1.5 with IC₅₀s of 46.6 ± 4.3 and 28.0 ± 2.3, 7.6 ± 0.7 and 6.2 ± 0.5, 153.5 ± 13.0 and 106.0 ± 4.7, 5.5 ± 0.5 and 4.8 ± 0.2 μM. Voltage dependences of activation (V_1/2) for hERG were statistically identical, 0.4 ± 0.8 mV and 2.5 ± 0.5 mV. In hNa_V1.5, the V_1/2 of inactivation and activation were statistically identical, −82.7 ± 0.1 mV versus − 84.9 ± 0.3 mV, −47.5 ± 0.3 mV versus − 45.0 ± 0.6 mV. Current density in both conditions in hERG experiments was similar, 47.0 ± 4.1 pA versus 42.3 ± 6.0 pA/pF.

Discussion

hERG and hNa_V1.5 screens run using frozen cells have statistically identical IC₅₀s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.     

A novel selective and orally bioavailable Nav1.8 channel blocker,PF-01247324, attenuates nociception and sensory neuron excitability

Background and Purpose

Na_V1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Na_v1.8 channel blocker of novel chemotype.

Experimental Approach

The inhibition of Na_v1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain.

Key Results

PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC₅₀: 331 nM) and in recombinantly expressed h Na_v1.8 channels (IC₅₀: 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNa_v1.5 channels (IC₅₀: ∼10 μM) and 65–100-fold selectivity over TTX-sensitive (TTX-S) channels (IC₅₀: ∼10–18 μM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC₅₀ 448 nM, and the block of both human recombinant Na_v1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models.

Conclusions and Implications

Using PF-01247324, we have confirmed a role for Na_v1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Na_v1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.     

Expression,Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties

The voltage-gated sodium channel Na_V1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of Na_V1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC₅₀ values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between Na_V1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through Na_V1.7 inhibition.     

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain

Loss-of-function mutations of Na_V1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na_V1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na_V1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na_V1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na_V1.7 inhibitors and significantly reduced in Na_V1.7^−/− mice. To validate the use of the model for profiling Na_V1.7 inhibitors, we determined the Na_V selectivity and tested the efficacy of the reported Na_V1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na_V1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na_V1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na_V channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na_V1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na_V1.7 inhibitors.     

Spider-venom peptides that target voltage-gated sodium channels: Pharmacological tools and potential therapeutic leads

Voltage-gated sodium (Na_V) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na_V channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na_V channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na_V channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na_V channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na_V channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na_V channels.     

Blocking effect of methylflavonolamine on human NaV1.5 channels expressed in Xenopus laevis oocytes and on sodium currents in rabbit ventricular myocytes

Aim:

To investigate the blocking effects of methylflavonolamine (MFA) on human Na_V1.5 channels expressed in Xenopus laevis oocytes and on sodium currents (I_Na) in rabbit ventricular myocytes.

Methods:

Human Na_V1.5 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. I_Na and action potentials in rabbit ventricular myocytes were studied using the whole-cell recording.

Results:

MFA and lidocaine inhibited human Na_V1.5 channels expressed in Xenopus oocytes in a positive rate-dependent and concentration-dependent manner, with IC₅₀ values of 72.61 μmol/L and 145.62 μmol/L, respectively. Both of them markedly shifted the steady-state activation curve of I_Na toward more positive potentials, shifted the steady-state inactivation curve of I_Na toward more negative potentials and postponed the recovery of the I_Na inactivation state. In rabbit ventricular myocytes, MFA inhibited I_Na with a shift in the steady-state inactivation curve toward more negative potentials, thereby postponing the recovery of the I_Na inactivation state. This shift was in a positive rate-dependent manner. Under current-clamp mode, MAF significantly decreased action potential amplitude (APA) and maximal depolarization velocity (V_max) and shortened action potential duration (APD), but did not alter the resting membrane potential (RMP). The demonstrated that the kinetics of sodium channel blockage by MFA resemble those of class I antiarrhythmic agents such as lidocaine.

Conclusion:

MFA protects the heart against arrhythmias by its blocking effect on sodium channels.     

Alcohol Withdrawal-Induced Seizure Susceptibility is Associated with an Upregulation of CaV1.3 Channels in the Rat Inferior Colliculus

Background:

We previously reported increased current density through L-type voltage-gated Ca²⁺ (Ca_V1) channels in inferior colliculus (IC) neurons during alcohol withdrawal. However, the molecular correlate of this increased Ca_V1 current is currently unknown.

Methods:

Rats received three daily doses of ethanol every 8 hours for 4 consecutive days; control rats received vehicle. The IC was dissected at various time intervals following alcohol withdrawal, and the mRNA and protein levels of the Ca_V1.3 and Ca_V1.2 α1 subunits were measured. In separate experiments, rats were tested for their susceptibility to alcohol withdrawal–induced seizures (AWS) 3, 24, and 48 hours after alcohol withdrawal.

Results:

In the alcohol-treated group, AWS were observed 24 hours after withdrawal; no seizures were observed at 3 or 48 hours. No seizures were observed at any time in the control-treated rats. Compared to control-treated rats, the mRNA level of the Ca_V1.3 α1 subunit was increased 1.4-fold, 1.9-fold, and 1.3-fold at 3, 24, and 48 hours, respectively. In contrast, the mRNA level of the Ca_V1.2 α1 subunit increased 1.5-fold and 1.4-fold at 24 and 48 hours, respectively. At 24 hours, Western blot analyses revealed that the levels of the Ca_V1.3 and Ca_V1.2 α1 subunits increased by 52% and 32%, respectively, 24 hours after alcohol withdrawal. In contrast, the Ca_V1.2 and Ca_V1.3 α1 subunits were not altered at either 3 or 48 hours during alcohol withdrawal.

Conclusions:

Expression of the Ca_V1.3 α1 subunit increased in parallel with AWS development, suggesting that altered L-type Ca_V1.3 channel expression is an important feature of AWS pathogenesis.