Selective Voltage-Gated Sodium Channel Peptide Toxins from Animal

Nov 21, 2017 - mutations are associated with many painful disorders.19 Local anesthetics such as lidocaine20 and antiepileptic drugs such as...
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Cite This: ACS Chem. Neurosci. 2018, 9, 187−197

Selective Voltage-Gated Sodium Channel Peptide Toxins from Animal Venom: Pharmacological Probes and Analgesic Drug Development Ying Wu,† Hui Ma,† Fan Zhang,* Chunlei Zhang, Xiaohan Zou, and Zhengyu Cao*

ACS Chem. Neurosci. 2018.9:187-197. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 01/15/19. For personal use only.

Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Development, China Pharmaceutical University, Nanjing 211198, China ABSTRACT: Voltage-gated sodium channels (Navs) play critical roles in action potential generation and propagation. Nav channelopathy as well as pathological sensitization contribute to allodynia and hyperalgesia. Recent evidence has demonstrated the significant roles of Nav subtypes (Nav1.3, 1.7, 1.8, and 1.9) in nociceptive transduction, and therefore these Navs may represent attractive targets for analgesic drug discovery. Animal toxins are structurally diverse peptides that are highly potent yet selective on ion channel subtypes and therefore represent valuable probes to elucidate the structures, gating properties, and cellular functions of ion channels. In this review, we summarize recent advances on peptide toxins from animal venom that selectively target Nav1.3, 1.7, 1.8, and 1.9, along with their potential in analgesic drug discovery. KEYWORDS: Voltage-gated sodium channels, pain, animal toxins, peptide therapeutic

1. VOLTAGE-GATED SODIUM CHANNEL STRUCTURE AND FUNCTION Voltage-gated sodium channels (Navs) are transmembrane proteins that conduct sodium ion (Na+) into cytosol upon activation. Navs play a central role in action potential (AP) generation and propagation in excitable cells, including cardiac myocytes, skeletal muscle cells, and neurons.1,2 Navs are also marginally expressed in nonexcitable cells, involved in noncanonical roles in regulating multiple pathophysiological functions.1,3 A Nav comprises a highly sequence-conserved α-subunit (220− 260 kDa) and one or two auxiliary β-subunits (30−40 kDa).4,5 The Nav α-subunit consists of four homologous domains (DI− DIV), each containing six transmembrane α-helixes (S1−S6) and a short membrane reentry loop between S5 and S6. The α-subunit comprises multiple domains, which are involved in pore-forming, voltage-sensing, and Na+ selectivity.6 The S4 transmembrane segment in each domain contains four to seven positively charged tri-amino acid repeats, in each of which an arginine or lysine is commonly followed by two hydrophobic amino acids. This positively charged motif in the S4 segment serves as the voltage sensor that moves outward to the extracellular part in a sliding-helix model upon depolarization.7 The movement of the S4 results in conformational change of the channel to an open state.6,7 Three hydrophobic amino acid residues, IFM, situated between transmembrane DIII-S6 and DIV-S1 form the inactivation gate, which quickly plugs into the pore and prevents additional Na+ influx.8,9 Four amino acids, DEKA, located between S5 and S6 form the selectivity filter for Na+ ions.9 A variety of toxins have been found to target Navs. Combined with radioligand binding, electrophysiological approach © 2017 American Chemical Society

and site-directed mutagenesis, at least seven distinct neurotoxin binding sites have been recognized in the Nav α-subunit (Table 1).10 According to their electrophysiological properties and binding sites, generally, Nav toxins can be classified into three groups: (1) pore-blocking toxins, which block Na+ conductance by interacting with neurotoxin site 1, including tetrodotoxin (TTX), μ-conotoxins, and saxitoxin (STX); (2) toxins that negatively shift the activation voltage and produce a persistent activation by binding to membrane-embedded neurotoxin site 2 (such as veratridine, grayanotoxin, and batrachotoxin) or site 5 (such as ciguatoxin and brevetoxin) and that prefer to interact with the open state of channel; (3) toxins that delay inactivation by binding to extracellular neurotoxin site 3, such as sea-anemone toxins and α-scorpion toxins. In addition to these well characterized toxins, some other toxins have distinct modes of action or neurotoxin binding sites. Spider toxins and β-scorpion toxins shift activation voltage to either depolarized or hyperpolarized direction by interacting with neurotoxin site 4. δ-Conotoxin prolongs channel inactivation, similar to α-scorpion toxins, by binding to neurotoxin site 6. Although the neurotoxin binding sites are topologically distinct, allosteric coupling has been demonstrated between sites 3 and 6 and between sites 2 and 5.11,12 In mammals, four β-subunits (β1−β4; encoded by SCN1B−4B genes) have been discovered. The β-subunits are type I transmembrane proteins, containing an extracellular signal peptide in the N-terminus, a transmembrane segment, and an immunoglobulin Received: October 26, 2017 Accepted: November 21, 2017 Published: November 21, 2017 187

DOI: 10.1021/acschemneuro.7b00406 ACS Chem. Neurosci. 2018, 9, 187−197

Review

ACS Chemical Neuroscience Table 1. Neurotoxins and Recognition Sites on Nav α-Subunit site

toxin

binding domains

1 2

tetrodotoxin, saxitoxin, μ-conotoxin batrachotoxin, veratridine, grayanotoxin

DI−IV SS1−SS2 DI DIV S6

3 4

α-scorpion toxins, sea anemone toxins, spider toxins β-scorpion toxins, spider toxins

5 6 7

brevetoxins, ciguatoxin δ-conotoxins pyrethroids

DI S5−S6, DIV S3−S4, S5−S6 DI S5−S6, DII S1−S2, S3−S4 DI S6, DIV S5 DIV S3−S4 DIII S6

effect

ref.

occlusion of the pore hyperpolarized shift in voltage-dependent activation, delayed inactivation destabilization of inactivated state

111,112 113−116

hyperpolarized shift in voltage-dependent activation, reduced current amplitude enhanced activation and delayed inactivation prolonged inactivation (subsite of site 3) inhibition of inactivation, shift in voltage-dependent activation

121,122

117−120

123 124 125

Table 2. Properties of Nav α-Subunitsa

a

types

gene symbol

Nav1.1

SCN1A

Nav1.2 Nav1.3

SCN2A SCN3A

Nav1.4

tissue distribution

TTXS/R

physiological function

effect of mutation

ref.

S S

AP initiation and repetitive firing in neurons; excitation-contraction coupling in cardiac myocytes AP initiation and conduction, repetitive firing AP initiation and conduction; repetitive firing

epilepsy, GEFS+, febrile seizures epilepsy, familial autism unclear

S

AP initiation and transmission in skeletal muscle

heart muscle

R

AP initiation and conduction

CNS, PNS, glia nodes of Ranvier PNS nociceptors

S

PNS (sensory neurons) PNS (DRG)

R

AP initiation and transmission in central neurons; partially responsible for the resurgent and persistent current in cerebellar Purkinje cells AP initiation and transmission in peripheral neurons; slow closedstate inactivation facilitates response to slow, small depolarizations contributes to action potential upstroke

myotonias, periodic paralysis, paramyotonia congenita long QT, cardiac conduction defects, Brugada syndrome cerebellar atrophy, ataxia, motor end plate disease IEM, PEPD, small fiber neuralgia, congenital indifference to pain painful peripheral neuropathy

70,71

R

modulates resting potential and amplifies small depolarizations

painful peripheral neuropathy

94,95

SCN4A

CNS, PNS, heart, DRG CNS CNS (embryonic) skeletal muscle

Nav1.5

SCN5A

Nav1.6

SCN8A

Nav1.7

SCN9A

Nav1.8

SCN10A

Nav1.9

SCN11A

S

S

126−128 129 14,27 130 131−133 134,135 21,40,41

+

AP, action potential; CNS, central nervous system; GEFS , genetic epilepsy with febrile seizures plus; IEM, inherited erythromelalgia; PEPD, paroxysmal extreme pain disorder; PNS, peripheral nervous system; TTX-S/R, tetrodotoxin-sensitive/resistant.

domain.9,13 Both excitable and nonexcitable cells have considerable amounts of β-subunit expression, which plays critical roles in modulating the localization, kinetics, and gating of Nav α-subunits.1,11,13,14 Based on similarity of their amino acid sequences, Navs can be classified into nine subtypes, Nav1.1−Nav1.9, encoded by genes SCN1A−5A and SCN8A−11A in mammals (Table 2).1,14 In addition to sequence homology, which is greater than 50% in identity, different subtypes of Navs display distinct gating kinetics. The nine subtypes can be categorized to be tetrodotoxin-sensitive (TTX-S, Nav1.1−1.4, Nav1.6, and Nav1.7) and tetrodotoxin-resistant (TTX-R, Nav1.5, Nav1.8, and Nav1.9) according to their sensitivities to TTX blocking. Nav expression is tissue and development dependent.2,11,15 Navs1.1−1.3 and 1.6 are primarily expressed in central nervous system (CNS). Navs1.4 and 1.5 are predominately expressed in skeletal and cardiac muscles, respectively. Navs1.7−1.9 are the major subtypes in peripheral nervous system (PNS).2

mechanical allodynia (pain hypersensitivity to normally innocuous stimuli).17 The molecular basis of chronic pain is complicated. Sensitization or changes in expression levels of ion channels, including Navs, contribute to chronic pain. Several sodium channel subtypes such as Navs1.3, 1.7, 1.8, and 1.9 are preferentially expressed in nociceptors where they propagate electric signals. These Nav subtypes play pivotal roles in the pathobiological progression of pain.18 Nav channelopathies derived from gene mutations are associated with many painful disorders.19 Local anesthetics such as lidocaine20 and antiepileptic drugs such as carbamazepine21 targeting Navs have been used for pain relief. However, these drugs provide limited analgesic efficacy in most cases due to lack of selectivity at inhibiting Navs including those expressed in brain and heart.22,23 Therefore, discovering Nav subtype selective inhibitors may provide promising analgesics with less side effects.

3. ANIMAL TOXINS TARGETING NAV SUBTYPES ASSOCIATED WITH PAIN Venoms from deadly venomous animals are a complex mixture containing a variety of peptides for defense and prey capture. Due to the critical roles of ion channels in the regulation of heartbeat and neuronal excitability,24 many peptide toxins from animal venoms have been demonstrated to modulate ion channel (including but not limited to sodium, potassium, and calcium channels) activities, often with exquisite potency and selectivity.25

2. NAVS INVOLVED IN PAIN Chronic pain affects approximately 20% of people worldwide.16 In general, chronic pain is categorized as neuropathic and nociceptive (nociceptor sensitization). Neuropathic pain, as a debilitating chronic pain condition, often follows nerve injury or malfunction derived from diseases such as cancer, diabetes mellitus, infection, autoimmune disease, and trauma. Clinical symptoms of neuropathic pain are characterized by hyperalgesia (increased pain perception of noxious stimuli) and 188

DOI: 10.1021/acschemneuro.7b00406 ACS Chem. Neurosci. 2018, 9, 187−197

Review

ACS Chemical Neuroscience Table 3. Primary Structures and Electrophysiological Properties of Nav1.3 Toxins from Animal Venoms toxin (species) μ-conotoxin BuIIIB (cone snail) BmK AS (scorpion) a

amino acid sequences

a

VGERCCKNGKRGCGRWCRDHSRCC#

major pharmacology, subtype selectivity blocks Nav1.3 (IC50 = 0.2 μM)

DNGYLLDKYTGCKVWCVINNESCNSECKIRGGYYGYCYFWKLACFCQGARKSELWNYNTNKCDGKL facilitates steady-state activation, inhibits slow inactivation and peak current of Nav1.3

ref 33,136 35,36

# indicates C-terminal amidation.

3.1. Nav1.3 and Animal Toxins. The expression of TTX-S Nav1.3 depends on the development stage. While the Nav1.3 expression level is significant in embryonic or neonatal dorsal root ganglion (DRG) neurons, adult sensory neurons have very low expression.26 Nav1.3 displays fast activation and inactivation kinetics and quick recovery from inactivation at membrane potentials less than −80 mV, suggesting a role of Nav1.3 in repetitive firing. Upon slow ramp depolarization, Nav1.3 produces large ramp currents.27 Nav1.3 contributes to accelerated repriming of TTX-S sodium currents within axotomized DRG neurons. This unique electrophysiological property may contribute to neuron development and altered neuronal excitability in injured neurons.18,27 In human injured neurons and painful neuromas, Nav1.3 expression, however, is significantly up-regulated.28−30 Antisense knock-down of Nav1.3 in nerve-injured rats attenuates neuropathic pain.31 However, controversy exists. Deletion of Nav1.3 either specifically from nociceptors or globally did not affect the abnormal discharges in injured nerves, nor did it alter the threshold of mechanical stimuli in neuropathic pain model suggesting a possible compensation mechanism in Nav1.3 knockout mice.32 Although controversy exists, Nav1.3 selective inhibitors are likely to be useful analgesics. Several Nav1.3 gating modifiers have been identified from animal venoms (Table 3). A peptide termed μ-conotoxin BuIIIB (μ-BuIIIB), discovered from the cone snail Conus bullatus, potently blocks Nav1.3 (IC50 = 0.2 μM),33 while another two structurally similar peptides, μ-KIIIA and μ-SIIIA are less potent (IC50 = 8 and 11 μM, respectively).34 Structure−activity relationship studies of μ-BuIIIB revealed the critical roles of C-terminal residues (Trp16, Arg18, and His20) for Nav inhibition.33 In addition to the C-terminal, the E3 to A replacement in the N-terminus of μ-BuIIIB significantly increased its potency. Another analog, ddSecBuIIIB, has been proven to display enhanced blockade to Nav1.3.33 However, whether the blockade of Nav1.3 by mutated μ-BuIIIB is useful in pain management needs further exploration. Indeed, ddSecBuIIIB also suppressed Nav1.4 activity, which is responsible for the initiation of excitation−contraction coupling in skeletal muscle cells. BmK AS, a long-chain neurotoxic polypeptide purified from scorpion Buthus martensi Karsch,35 has been reported to suppress Nav1.3 peak currents at nanomolar concentrations and shift the voltage-dependent activation and inactivation to the hyperpolarized direction by modulating neurotoxin site 4.36 In rodent pain models, BmK AS produced significant analgesic effects on both inflammation-induced spontaneous pain and mechanical hyperalgesia.37 Although several toxins have been reported to be Nav1.3 selective, little in vivo information was available in the literature. The role of Nav1.3 on pain maintenance and/or progression

remains unclear. Nevertheless, these discovered Nav1.3 selective peptide inhibitors may represent valuable tools to evaluate whether Nav1.3 can serve as a drug target for chronic pain management. 3.2. Nav1.7 and Animal Toxins. TTX-S Nav1.7 expression is mainly found in the peripheral nociceptors.15,22 The kinetics of Nav1.7 activation and inactivation are quick and the closed-state inactivation is slow allowing it to amplify subthreshold depolarizations.18 Nav1.7 is therefore considered to be a threshold channel producing substantial ramp currents for firing APs.38 Nav1.7 also produces resurgent currents upon repolarization following a strong depolarization.39 The expression profile, together with its unique property in the actin potential generation, suggested that Nav1.7 may serve as a promising drug target for pain drug development.18,39 Genetic evidence has further demonstrated the role of Nav1.7 in pain onset and maintenance in humans and other mammals.39 In humans, gain-of-function mutations of Nav1.7 have been demonstrated to be the etiology of several painful disorders such as paroxysmal extreme pain disorder (PEPD),21 small fiber neuralgia (SFN),15 and inherited erythromelalgia (IEM).40 These mutated Nav1.7 channels displayed enhanced Na+ currents and lowered threshold for activation, which resulted in increased firing frequency in response to graded suprathreshold stimuli,39 thereby enhancing hyperexcitability in DRG neurons. By contrast, Nav1.7 loss-of-function mutations are associated with congenital indifference to pain (CIP).41 Importantly, CIP patients have no aberrant cognitive and motor development.39 In addition, manipulation of Nav1.7 expression in rodent models also supports the notion. Global knock-down of mouse Nav1.7 ameliorated diabetic pain42 and thermal hyperalgesia induced by complete Freund’s adjuvant administration.43 Conditional knockout of Nav1.7 in DRG neurons normalized inflammatory pain and thermal hyperalgesia induced by burn injury without affecting neuropathic pain.39,44 DRG neurons from Nav1.7 knockout mice displayed similar peak amplitude of AP; however, the rising phase was slowed. A population of 30% of DRG neurons from Nav1.7 knockout animals failed to generate APs.45 Therefore, Nav1.7 is one of the promising drug targets for analgesic development with broad interests.15,46 Many selective Nav1.7 toxins have been reported (Table 4). μ-SLPTX-Ssm6a, a peptide containing 46 amino acid residues with three disulfide bonds, has been isolated from the centipede Scolopendra subspinipes mutilans. μ-SLPTX-Ssm6a potently inhibited Nav1.7 (IC50 = 25 nM) by shifting voltage-dependent activation to more depolarized potentials, which is 150-fold more potent than its effect on other Navs except for Nav1.2, which is 32-fold.47 In thermal- and acid-induced pain models, μ-SLPTX-Ssm6a produced comparable analgesic effect to morphine without adverse effects on cardiovascular system and CNS.15,47 189

DOI: 10.1021/acschemneuro.7b00406 ACS Chem. Neurosci. 2018, 9, 187−197

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ACS Chemical Neuroscience Table 4. Primary Structures and Electrophysiological Properties of Nav1.7 Toxins from Animal Venoms toxin (species) ProTx-II (tarantula) CcoTx1 (tarantula) μ-TRTX-Phlo1a (tarantula) μ-TRTX-Phlo1b (tarantula) huwentoxin-IV (spider) GpTx-1 (tarantula) hainantoxin-IV (spider) hainantoxin-III (spider) μ-SLPTX-Ssm6a (centipede) μ-conotoxin PIIIA (cone snail) μ-conotoxin KIIIA (cone snail) a

amino acid sequencesa

major pharmacology, subtype selectivity

ref. 48,83,137

DCLGWFKSCDPKNDKCCKNYTCSRRDRWCKYDL

inhibits Na+ conductance and shifts activation to depolarizing direction (IC50 = 0.3 nM, >100-fold selectivity) blocks current, shifts activation to depolarizing direction

52

ACRELLGGCSKDSDCCAHLECRKKWPYHCVWDWTI#

shifts activation to depolarizing direction (IC50 = 459 nM)

61

ACRELLGGCSKDSDCCAHLECRKKWPYHCVWDWTF#

shifts activation to depolarizing direction (IC50 = 360 nM)

61

ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQI#

blocks current hNav1.7 (IC50 = 17 nM) > rNav1.2 > rNav1.3 ≫ hNav1.5 > rNav1.4 Nav1.7 (IC50 = 10 nM) > Nav1.4 ≫ Nav1.5

53,54

YCQKWMWTCDSERKCCEGMVCRLWCKKKLW

DCLGFMRKCIPDNDKCCRPNLVCSRTHKWCKYVF#

ZRLCCGFOKSCRSRQCKOHRCC#

gating modifier hNav1.7 (IC50 ≈ 21 nM) > rNav1.2 > rNav1.3 ≫ hNav1.5 > rNav1.4 binds to site 4 in the closed state Nav1.7 (IC50 = 232 nM) > Nav1.2 > Nav1.3 ≫ Nav1.5 > Nav1.4 shifts activation to depolarizing direction hNav1.7 (IC50 = 25 nM) > hNav1.2 > hNav1.1 ≫ hNav1.3, 1.4, 1.5 blocks current Nav1.4 (IC50 = 41 nM) ≫ hNav1.7 (3.1 μM)

CCNCSSKWCRDHSRCC#

blocks current rNav1.2 > rNav1.4 > rNav1.7 ≫ rNav1.5

ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI# GCKGFGDSCTPGKNECCPNYACSSKHKWCKVYL# ADNKCENSLRREIACGQCRDKVKTDGYFYECCTSDSTFKKCQDLLH

50 57,58 60 47 62,63

64

# indicates C-terminal amidation; Z, pyroglutamate; O, 4-hydroxyproline.

HWTX-IV bound to neurotoxin site 4 of hNav1.7 in the closed state and trapped the voltage sensor in its inward position. A triple mutant, E1G,E4G,Y33W-HwTx-IV, displayed a 42-fold (IC50 = 0.4 ± 0.1 nM) selectivity for Nav1.7 over other Navs.53,54 HWTX-IV showed antinociceptive effects in murine neuropathic pain models. The analgesic effect of HWTX-IV in spinal nerve injury (SNI) model lasted longer and displayed higher efficacy than mexiletine.55 Hainantoxin (HNTX)-IV (also termed β-TRTX-Hn2a), a typical cystine knot peptide purified from Ornithoctonus hainana, selectively inhibited TTX-S, but not TTX-R Na+ currents.56,57 HNTX-IV preferentially blocked hNav1.7 (IC50 ≈ 21 nM) with little effect on rNav1.4 and hNav1.5. HNTX-IV also blocked the rNav1.2 and rNav1.3 (IC50 = 36 and 375 nM, respectively).58 HNTX-IV modification of Nav1.7 was dependent on depolarization potentials. At depolarization potentials below +70 mV, HNTX-IV suppressed Nav1.7 activity. However, partial agonism was observed at depolarization potentials above +70 mV.58 Nevertheless, in physiological and pathological conditions, no evidence shows that the membrane potential can reach +70 mV. HNTX-IV administration alleviated acute inflammatory pain and SNI-induced neuropathic pain.59 HNTX-III, another toxin from the spider Ornithoctonus hainana, inhibited Nav1.7, Nav1.2, and Nav1.3 with similar potency, although the selectivity against Nav1.4 and 1.5 was over 100-fold. Although it was bound to neurotoxin site 4, the mode of action of HNTX-III was distinct from β-scorpion toxins and other β-spider toxins. HNTX-III trapped the domain II voltage sensor in the closed state and therefore suppressed the Nav1.7 Na+ current.60 The in vivo analgesic data for HNTX-III are not available. Novel peptides μ-TRTX-Phlo1a, -Phlo1b, and -Phlo2a, from Australian Phlogius sp. tarantula, modified hNav1.7 at concentrations ranging from high nanomolar to sub-micromolar. Phlo1a and Phlo1b both contain 35 amino acid residues with only one residue difference. Phlo1a and Phlo1b belong to NaSpTx family 2 with marginal inhibition on Nav1.2 and Nav1.5 and therefore are selective Nav1.7 inhibitors. Phlo2a was less selective, as it robustly inhibited rNav1.2 and hNav1.5 as well at similar

In addition to centipede toxin, several spider toxins have been identified to potently and selectively inhibit Nav1.7.15 The best-known example is protoxin II (ProTx-II), isolated from Thrixopelma pruriens. The IC50 value (0.3 nM) for ProTx-II inhibiting Nav1.7 was ∼100-fold more potent than that on other Nav subtypes.48 ProTx-II inhibited Nav1.7 peak current by shifting the activation potentials to depolarized direction. It is interesting that local or systematic administrations of ProTx-II failed to produce analgesic effect in an inflammatory pain model. The explanation that ProTxII lacks analgesic effect because of its inability to penetrate blood−brain barrier48 is unlikely to be true since Nav1.7 is predominantly expressed in peripheral nociceptors. Nevertheless, the selectivity on Nav1.7 of this peptide does provide a valuable tool to study the cellular function of Nav1.7.45,48 GpTx-1, a 34-residue, C-terminal amidated peptide toxin with three-disulfide bounds, was reported to inhibit Nav 1.7 with less selectivity. Using multiattribute positional scan analoging, an engineered GpTx-1 analog was reported to inhibit Nav1.7 with IC50 values of ∼1.6 nM, which was over 1000-fold more potent than that on Nav1.4 and Nav1.5.49,50 Local administration of GpTx-1 significantly reduced the pain behavior induced by scorpion toxin OD1. However, systemically delivered GpTx-1 had no efficacy in rodent pain models.51 Recently, Shcherbatko et al. engineered a potent and selective Nav1.7 inhibitor using the template, tarantula ceratotoxin-1 (CcoTx1), a Nav toxin isolated from Ceratogyrus cornuatus. The CcoTx1 was further modified to achieve more potent and selective microproteins using semirational approaches. The microprotein suppressed Nav1.7 with an IC50 value of 2.5 nM, over 1000-fold selectivity on Nav1.4 and Nav1.5, 80- and 20-fold selectivity on Nav1.2 and Nav1.6, respectively.52 However, no in vivo data were available regarding the efficacy of the microprotein in pain models. Huwentoxin (HWTX)-IV, a 35-residue peptide purified from the spider Selenocosmia huwena, containing three disulfide bridges, was reported to inhibit hNav1.7 (IC50 = 17 ± 2 nM) without effects on Nav1.5 even at a concentration of 10 μM. 190

DOI: 10.1021/acschemneuro.7b00406 ACS Chem. Neurosci. 2018, 9, 187−197

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191

a

# indicates C-terminal amidation; Z, pyroglutamate; O, 4-hydroxyproline.

105,140 dramatic facilitation of rNav1.9 currents while only modest inhibition of rNav1.8 at 100 nM KEGYLMDHEGCKLSCFIRPSGYCGRECGIKKGSSGYCAWPACYCYGLPNWVKVWDRATNKC#

139

ZRCCNGRRGCSSRWCRDHSRCC#

μ-conotoxin SmIIIA (cone snail) μ-conotoxin TsIIIA (cone snail) TsVII (scorpion)

blocks TTX-R Na+ current in rat DRG (IC50 = 2.6 μM)

GTACSCGNSKGIYWFYRPSCPTDRGYTGSCRYFLGTCCTPAD

GCCRWPCPSRCGMARCCSS

138

82

83

APETx2 (sea anemone)

RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV

ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS

shifts activation to depolarizing direction Nav1.2 ∼ Nav1.5 ∼ Nav1.7 ∼ Nav1.8 blocks current and shifts activation to depolarizing direction rNav1.8 (IC50 = 2.6 μM) > TTX-S Nav blocks TTX-R Na+ current

79

blocks current hNav1.8 (IC50 = 102 nM) > rNav1.2, rNav1.3, hNav1.5, hNav1.7 ≫ rNav1.9 blocks current Nav1.4 > rNav1.8 (IC50 = 95.9 nM) > Nav1.5 ACSKKWEYCIVPILGFVYCCPGLICGPFVCV

80,81

gating modifier TTX-R Nav (rat DRG) (IC50 = 82.8 nM) ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV

μO-conotoxin MrVIA (cone snail) μO-conotoxin MrVIB (cone snail) μO-conotoxin MfVIA (cone snail) ProTx-I (tarantula)

amino acid sequencesa toxin (species)

Table 5. Primary Structures and Electrophysiological Properties of Nav1.8 and Nav1.9 Toxins from Animal Venoms

major pharmacology, subtype selectivity

ref.

concentration range. Although the selectivities on Nav1.7 are different, these three peptides all shifted activation voltage to depolarized direction rendering Nav1.7 more difficult to open.61 Other species like cone snails also contain a diverse range of Nav1.7 inhibitors; however, their selectivities on Nav1.7 were relatively poor. μ-Conotoxin PIIIA, isolated from Conus purpurascens, is a hNav1.7 inhibitor with an IC50 value of 3.1 μM. μ-Conotoxin PIIIA also blocked Nav1.4.62,63 Another Nav1.7 inhibitor, μ-conotoxin KIIIA, isolated from Conus kinoshitai, suppressed Nav1.7, Nav1.2, and Nav1.4 at comparable concentration ranges. Nevertheless, μ-conotoxin KIIIA significantly attenuated mouse pain behaviors induced by formalin, most likely through its action on Nav1.7.64,65 In conclusion, venom-derived peptides have provided a broad range of Nav1.7 inhibitors, most of which displayed significant analgesic effect in murine pain models. Further investigation on structure−activity relationships in combination with point mutation and electrophysiology may provide highly potent yet selective Nav1.7 inhibitors with metabolic stability for analgesic development targeting Nav1.7. 3.3. Nav1.8 and Animal Toxins. Similar to Nav1.7, TTX-R Nav1.8 is preferentially expressed in PNS nociceptors including small-diameter DRG neurons and nodose and trigeminal ganglia neurons.66 Nav1.8 displays fast activation, slow inactivation, and rapid repriming kinetics.66,67 Nav1.8 is responsible for the majority of inward Na+ current contributing to the upstroke of neuronal APs, supporting the repetitive firing in depolarized neurons.22,68 Nav1.8 is also responsible for subthreshold membrane oscillations,69 which contribute significantly to hyperexcitability following injury of sensory neurons. The fundamental neurophysiological role of Nav1.8 in nociceptive neurons suggests that Nav1.8 has significant effect on nociceptor excitability, thus linking to pain management in humans. Although genetic loss-of-function mutations were not yet described in humans, gain-of-function mutations of Nav1.8 were shown to produce hyperexcitability and inappropriate spontaneous firing in neurons. These gain-of-function mutations were thought to be the etiology of painful peripheral neuropathy observed in humans.70,71 Nav1.8 has been confirmed to be involved in cold, inflammatory, and neuropathic pain in knockout67,72 and knock-down73,74 murine models. Bierhaus et al. reported that methylglyoxal modification of Nav1.8 increased nociceptor firing, contributing to hyperalgesia in mouse diabetic neuropathy. These studies provided evidence for a previously unidentified pathway in which methylglyoxal directly induced hyperalgesia and may suggest that Nav1.8 was a valid therapeutic target for diabetic pain.75 Moreover, some small molecular inhibitors, such as A-803467 and PF-01247324, which are potent and selective Nav1.8 blockers, were reported to produce significant antinociceptive effect in neuropathic and inflammatory pain models.76,77 Overall, although the relationship between Nav1.8 expression and neuropathic signaling is not as clear as that of Nav1.7, electrophysiological properties, together with its tissue distribution, suggest that Nav1.8 is crucial in nociceptive signaling. To date, several selective Nav1.8 inhibitors have been identified from animal venom (Table 5). The μO-conotoxin MrVIA from freshwater snail Conus marmoreus was reported to block TTX-R Na+ current with an IC50 value of 82.8 nM, which was 10-fold more potent than that on TTX-S Na+ currents in DRG neurons.78 The inhibition of Na+ current was voltage-dependent. The inhibition was significantly diminished with more depolarized voltage steps.79 The other μO-conotoxin, MrVIB,

78,79

ACS Chemical Neuroscience

DOI: 10.1021/acschemneuro.7b00406 ACS Chem. Neurosci. 2018, 9, 187−197

Review

ACS Chemical Neuroscience

induce pain.93−95 However, a specific de novo gain-of-function mutation (L899P) of Nav1.9 was identified in patients unable to experience pain.96 In addition, upregulated mRNA and protein levels of Nav1.9 were observed in DRG neurons from diabetic neuropathic rats.97 Interestingly, levels of Nav1.9 mRNA and protein as well as the current density were downregulated in other neuropathic pain models.98−100 Nav1.9 is hypersensitive under inflammatory conditions on peripheral nociceptor terminals and therefore contributes to peripheral sensitization.101 Inflammatory mediators such as interleukins (ILs) were reported to sensitize Nav1.9, which contributes to AP generation, and increased AP frequency upon depolarizing stimuli.102,103 Nav1.9 knockout mice showed attenuated somatic inflammatory hyperalgesia in response to inflammatory mediators.101,104 Taken together, the role of Nav1.9 in inflammatory pain was much clearer compared to neuropathic pain. Heterologous expression of Nav1.9 was difficult, which limited the investigation of its functional properties and the screening of Nav1.9 modulators, including animal toxins.105 Therefore, although an increasing number of peptide toxins are valuable to elucidate the role of Navs in neuron excitability, discovery of selective Nav1.9 toxins is difficult. TsVII and ProTx-I were proposed to interact with paddle motifs of Nav1.9 in DRG neurons without subtype-selectivity.105 μ-Conotoxin, SIIIA selectively inhibited TTX-R rather than TTX-S Na+ currents in dissociated rat adult small-diameter DRG neurons and produced analgesic activity in rodent hyperalgesia models.106,107 Thus, the pharmacological tools for selective inhibition of Nav1.9 are lacking. Recently established HEK-293 cells and ND7/23 cells that heterologously express Nav1.9108,109 can hopefully facilitate identification of selective Nav1.9 modulators. Due to less similarity in the primary structure of Nav1.9 to other Nav subtypes, discovery of a selective Nav1.9 inhibitor may be easier. The preferential distribution of Nav1.9 within nociceptors suggests that selective Nav1.9 inhibitor might be a favorable pain target, at least for inflammatory pain, with minimal cognitive or cardiac adverse side effects.

displayed 10-fold selectivity in inhibiting TTX-R Na+ currents than for TTX-S Na+ current in nociceptive DRG neurons. Further studies demonstrated that MrVIB blocked human Nav1.8 with 10-fold selectivity to other Navs.80 MrVIB displayed efficacious long-lasting analgesic effects in animal models of persistent pain.80,81 MrVIB (0.03−3 nmol) intrathecal administration significantly reduced mechanical allodynia and hyperalgesia although motor activity deficits were observed at a much higher dose.80 Thus, MrVIB may represent a promising lead compound to develop Nav1.8-selective inhibitor for treating inflammatory and neuropathic chronic pain. Another μO-conotoxin, MfVIA, a hydrophobic 32-residue peptide, isolated from Conus magnif icus, potently inhibited Nav1.4 and Nav1.8 with much lower affinity to other Navs.79 However, there are no in vivo data available due to its action on Nav1.4. APETx2, a toxin from the sea anemone Anthopleura elegantissima, was reported to be an acid-sensing ion channel 3 (ASIC3) modulator. APETx2 rationalized inflammatory and acid-induced pain was initially proposed to be through inhibiting ASIC3. Recent studies demonstrated that APETx2 also inhibited the TTX-R Nav1.8 Na+ currents in DRG neurons (IC50 = 2.6 μM) with marginal effect on TTX-S Na+ currents. APETx2 shifted activation voltage of Nav1.8 rightward therefore reducing the maximal macroscopic conductance. Thus, in vivo analgesic effect of APETx2 was possible through inhibition of both Nav1.8 and ASIC3 channels, two pain promoting targets.82 ProTx-I, identified from tarantula Thrixopelma pruriens, was the first highly potent TTX-R Nav1.8 inhibitor, but with poor selectivity.83 Many spider toxins have been demonstrated to inhibit Nav1.8 currents with low affinity or without selectivity. Ceratotoxins (CcoTx)1−3 and phrixotoxin 3 (PaurTx3) inhibited Nav1.8 Na+ current at micromolar concentrations.84 JZTX-V85 and JZTX-IX86 inhibited rat Nav1.8 without selectivity.87 Recently discovered μ-TRTX-Hl1a from Haplopelma lividum appeared to suppress TTX-R Na+ current rather than TTX-S Na+ current suggesting the selectivity of μ-TRTX-Hl1a on Nav1.8. The comparable analgesic effect of μ-TRTX-Hl1a to morphine in both neuropathic and inflammatory pain suggests its usefulness in pain drug discovery.88 As described above, the search for Nav1.8 selective inhibitors is relatively lagging when compared to Nav1.7. Nevertheless, genetic evidence and pharmacological data all suggest the potential of Nav1.8 selective inhibitors for pain drug development. 3.4. Nav1.9 and Animal Toxins. TTX-R Nav1.9 is preferentially expressed in intrinsic myenteric sensory nociceptors, small-diameter (