Voltage-Gated Sodium Channels: Structure, Function, Pharmacology

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Voltage-Gated Sodium Channels: Structure, Function, Pharmacology and Clinical Indications Manuel de Lera Ruiz, and Richard L. Kraus J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 Apr 2015 Downloaded from http://pubs.acs.org on May 1, 2015

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Journal of Medicinal Chemistry

Voltage-Gated Sodium Channels: Structure, Function, Pharmacology and Clinical Indications Manuel de Lera Ruiz,* and Richard L. Kraus Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, United States

Abstract: The tremendous therapeutic potential of voltage-gated sodium channels (Navs) has been the subject of many studies in the past and is of intense interest today. Nav1.7 channels in particular have received much attention recently because of strong genetic validation of their involvement in nociception. Here we summarize the current status of research in the Nav field and present the most relevant recent developments with respect to the molecular structure, general physiology, and pharmacology of distinct Nav channel subtypes. We discuss Nav channel ligands such as small molecules, toxins isolated from animal venoms and the recently identified Nav1.7-selective antibody. Furthermore, we review eight characterized ligand binding sites on the Nav channel α subunit. Finally, we examine possible therapeutic applications of Nav ligands and provide an update on current clinical studies.

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INTRODUCTION Voltage-gated ion channels (VGICs) are transmembrane proteins that play important roles in the electrical signaling of cells. The activity of VGICs is regulated by the membrane potential of a cell and open channels allow the movement of ions along an electrochemical gradient across cellular membranes. Depending on the ions conducted VGICs can be classified as voltage –gated sodium,1 potassium, calcium, or chloride channels. Voltage-gated sodium currents were discovered by Hodgkin and Huxley in 1952 when studying the electric conductance in axons of giant squids.2 Twenty six years later Nav channels (Navs) were isolated and purified from the eel electroplax.3 To date, nine Nav1 channel subtypes named Nav1.1 through Nav1.9 have been cloned. A novel family of Nav2 channels was discovered recently, however, little is known about its function.4 Mammalian Nav channels are formed by a large pseudo-tetrameric pore-forming α subunit (260 kDa) that associates with one or two β subunits (30-40 kDa). In contrast, prokaryotic Nav channels that have been used to explore the structure of eukaryotic Navs are formed by homotetramers. Nav channel α subunits show tissue specific expression profiles. Nav1.1, Nav1.2, and Nav1.3 subtypes are expressed in the central nervous system (CNS). Nav1.6 is expressed in both the peripheral and central nervous system, whereas Nav1.7, Nav1.8, and Nav1.9 are mostly restricted to the peripheral nervous system (PNS).5 Nav1.4 and Nav1.5 channels are abundant in skeletal and cardiac muscles, respectively. Nav channels are responsible for generating the Na+ currents underlying the initiation and propagation of action potentials in nerves and muscle fibers. In primary sensory neurons, for example, the depolarization of an axon by a noxious stimulus, leads to the transmission of sensory information through the nervous system to the brain which may be perceived as pain. In skeletal and cardiac muscle cells, received action potentials produce muscle contraction enabling body movements and blood flow. 2 ACS Paragon Plus Environment

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Interestingly, Nav channels are the molecular targets for a broad range of natural neurotoxins, such as tetrodotoxin (TTX, 1, Figures 3A and 6), saxitoxin (STX, 2, Figure 6), and batrachotoxin (BTX, 11, Figures 3B and 8), as well as peptide toxins isolated from the venoms of scorpions, spiders, sea anemones, and cone snails (such as those in Figures 3C, 3D, 3E and 3F). Those toxins interact with at least six known receptor sites, named Site 1-6, and inhibit or modulate the gating properties of Nav channels.6 The high degree of amino acid sequence homology among the different Nav subtypes makes finding subtype-selective ligands extremely difficult. All Nav subtypes known to date can be classified by their sensitivity to the guanidine-based neurotoxin TTX, a toxin isolated from the puffer fish. Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, and Nav1.7 are blocked by low nanomolar concentrations of TTX; therefore these subtypes are classified as TTX-sensitive, whereas, Nav1.5, Nav1.8, and Nav1.9 are inhibited by only high micromolar TTX concentrations and are considered TTX-resistant channels. Nav channels are very dynamic structures. There are three primary states for Nav channels: a closed resting state, an open conducting state, and a non-conducting inactivated state. Many known natural and synthetic Nav ligands display different affinities to distinct ion channel states, a phenomenon called statedependence. In contrast, other ion channel modulators show little preference for a particular channel state in their interactions, and are considered state-independent modulators. The role of Nav channels in neuronal and cardiac disorders has long been known and non-selective sodium channel blockers have been developed as anticonvulsant, antiarrhythmic, and local anesthetic drugs in the past. More recently mutations in human genes encoding Nav α channel subtypes have been linked to channelopathies, such as epilepsy, cardiac arrhythmias, and chronic pain syndromes. The analysis of numerous gain and loss of function mutations have revealed invaluable information about the physiological role of Nav channels in disease. Currently



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enormous efforts are underway to find and study the effects of subtype-selective Nav ligands in preclinical disease models and eventually in the clinic. The wealth of publications and patents in recent years reflects the high interest in this research field. In 2013 approximately, 1,500 articles on Navs were published, followed by a further 1,300 in 2014. Among these, approximately 200 articles on the Nav1.7 subtype were published in 2013, and 160 in 2014.7 From this enormous volume of literature, we will attempt to extract and summarize the most relevant information and developments in this review, focusing on channel structure, function and role of Navs in diseases. In addition, we will discuss the current status of the most significant natural, semisynthetic, and artificial ligands of Nav channels and provide an update on recent clinical developments.

MOLECULAR ARCHITECTURE Just over 30 years ago, Catterall, Beneski, and Hartshorne proposed the basic structure of Nav channels based on covalently labeling protein components of purified rat brain Navs with a photoactive derivative of a scorpion toxin.8 Many other achievements and advances in Nav channel structure research followed. Of paramount importance is the complete sequencing of the human genome, which has provided us with the identification of 143 ion channel proteins. Furthermore, the recent X-ray crystal structure determination of bacterial Nav channels (NavAb, NavRh, and NavMs from Arcobacter butzleri, Alphaproteobacterium HIMB114 and Magnetococcus sp, respectively) has allowed us to visualize these channels three-dimensionally in different functional states such as closed,9 potentially inactive,10,11 and open.12 These studies made it possible to combine human Nav channel sequences and bacterial crystallographic data to build homology models. 1,13 Although these homology models coupled with molecular dynamics


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simulations (MDS) have provided insight into how these channels function at the molecular level, unanswered questions still remain, mainly due to the conformational differences between the stabilized nature of the crystallized receptor and the dynamic nature of the wild-type channel. Numerous studies using prokaryotic Nav channels have provided us with insight into the structure and function of bacterial Nav channels, translatable to eukaryotic Nav channels.14 Doubtless, an X-ray crystal structure of an eukaryotic Nav channel, which has yet to be solved, will provide us with a further understanding of how eukaryotic Navs function at the molecular level.

Molecular architecture of α subunit. As mentioned in the introduction, eukaryotic Nav channels consist of an α subunit, which can be coupled to one or two β subunits. In humans there are nine different α subunits Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9, encoded by the genes SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A, respectively. The different α subunits define the distinct Nav channel subtypes and contain the receptor sites for drugs and toxins that act on Nav channels. The α subunits are large, single-chain polypeptides composed of approximately 2,000 amino acid residues organized in four homologous domains, designated DI to DIV, that form a pseudotetrameric structure (Figure 1). The sequence homology of mammalian Nav subtypes is very high, being greater than 50% in the transmembrane and extracellular domains.5a Each domain is composed of six transmembrane helical segments named S1 to S6. In contrast, prokaryotic Nav channels are far simpler, consisting of homotetramers of four identical polypeptide chains, each one also containing six transmembrane segments (S1-S6) and exhibits high sequence homology when compared to each individual eukaryotic domain.



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Voltage-sensing domain (VSD). Segments S1-4 of Nav channel α subunits form the VSD, an important structural module whose function is the regulation of channel opening upon depolarization of the membrane. The VSD is relatively flexible and is comprised of the S1–S4 segments of each domain. This flexibility is primarily mediated by movement of positively charged arginine and lysine residues positioned at every third residue within each S4 helix. The four voltage-sensing domains are arranged around a central aqueous channel formed by the pore domain (PD). The VSD is connected to the PD by an intracellular linker between transmembrane segments S4 and S5. Upon depolarization the positively charged S4 transmembrane segments are believed to move toward the extracellular surface. This motion is transferred to the pore domain via intracellular linkers causing a conformational change that results in the opening of the channel pore. During depolarization the channel inactivates as the inactivation gate (vide infra) folds into the channel pore. Upon membrane repolarization Nav channels recover from inactivation and the S4 segments return to their resting positions becoming available for the next depolarization. From a study of the functional contributions of the amino acid residues at the VSD of skeletal muscle channel subtype Nav1.4, Ahern and coworkers found that the highly conserved aromatic side chain at the S2 hydrophobic core makes distinct functional contributions in each of the four Nav domains. This study showed that surprisingly, not all of the four S1-S4 structures contributing to the VSD adopt the same structural conformation at a specific Nav channel state.15

Pore domain (PD). Segments S5, S6 and the extracellular connecting pore-loops (Ploops) form the channel pore and the selectivity filter (SF). As will be discussed later in this review, most Nav-blocking drugs bind to residues at the central water-filled cavity of the pore. Recent structural Nav studies have revealed the existence of additional lateral lipid-filled 6 ACS Paragon Plus Environment

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openings, called fenestrations, connecting the exterior of the channel protein with the central pore. Nav channel fenestrations could create pathways for small hydrophobic Nav-blockers to access the pore from the side through the membrane. Kaczmarski, J. A. and Corry, B. have investigated Nav subtype specific factors contributing to the size and dynamics of Nav channel fenestrations using MDS and several bacterial and eukaryotic Nav models.16 Interestingly, although the VSD is a critical part in the structure of Navs, bacterial Nav pore-only constructs lacking the VSD are functional and selective ion channels.17 Recently, Shaya and coworkers have published the X-ray crystal structure of a closed conformation of NavAe1p, a pore-only prokaryotic Nav channel.18 This structure reveals a PD in which the pore-lining S6 helix connects to a helical cytoplasmic tail and an S6 activation gate residue is a critical part of its gating mechanism. Surprisingly, the structure also has an outer ion site in the selectivity filter (vide infra) that suggests the presence of multiple ion-binding sites and the possibility that various ions may occupy the selectivity filter simultaneously.

Selectivity filter (SF). The PD includes the selectivity filter (SF, Figure 1C), the narrowest part of the pore that distinguish ions with similar charges and radii. The single polypeptide chain of the eukaryotic α subunit allows an asymmetrical distribution of amino acid residues lining the SF. The SF is invariantly composed of aspartate (D) in DI, glutamate (E) in DII, lysine (K) in DIII, and alanine (A) in DIV (or DEKA) forming the geometrically narrowest region of the ion pore, which is called the constriction site or inner ring and selectively allows the flux of hydrated Na+ through the ion pore. A salt bridge between lysine and aspartate or glutamate is responsible for a pore size that accommodates Na+ better than K+, although different profiles in the free energy of these ions (derived from MDS and hydration numbers) can also be responsible for the ion selectivity. Researchers have shown that replacing lysine at position 1,237 by glutamate 7 ACS Paragon Plus Environment


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abolishes Na+ selectivity, making the channel permeable to Na+, K+, Ca2+, and Ba2+.19 Closer to the extracellular region there is another ring formed by two glutamate and two aspartate residues (EEDD) and referred to as the outer ring, which also plays an important role in Na+ permeation. Gong and coworkers have recently analyzed the mechanism of Na+/K+ selectivity in mammalian sodium channels using MDS, and a homology model where the four serine (S180) residues of the constriction site of bacterial NavRh were mutated to DEKA to mimic the SF of mammalian Nav channels. These studies showed that the lysine residue screens Na+ and electrostatically repels it to a highly Na+-selective location formed by the clustered carboxylate groups of aspartate and glutamate.20 In collaboration with Harvard Medical School, Gong’s group has published a study where, after combining structural biology with MDS, they have identified two Na+ binding sites at the bacterial NavRh SF. Surprisingly, it was observed that Na+ translocates in an asymmetrical manner, up to 5Å off the central axis of the permeation pathway. This is due to the fact that the conformations of the functional groups of the amino acids that constitute the SF are not identical adopting an asymmetric tetramer. This observation suggests that a similar asymmetric phenomena may occur in eukaryotic counterparts where the level of asymmetry between the four domains is higher.21

Inactivation gate. Another key structural element in the α subunit is the inactivation gate (Figure 1A), which consists of an intracellular loop connecting the homologous domains DIII and DIV. The inactivation gate acts as a hinged lid and folds into the intracellular mouth of the pore during fast inactivation.22

Activation gate. Four hydrophobic amino acid residues, one from each intracellular end of the S6 segments, form a small intracellular cavity named the activation gate (Figure 1C). The


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putative activation gate in wild-type Nav1.7 channels consists of four aromatic residues, one from each S6 helix (Y405 in DI, F960 in DII, F1449 in DIII, and F1752 in DIV). Using structural modeling–guided mutagenesis, Waxman and colleagues have demonstrated that when the L955 residue in DIVS6 is deleted, the orientation of F960 shifts radially toward the S6 of DIII disrupting the activation gate.23 Deletion of L955 of human Nav1.7 channels from a family with inherited erythromelalgia causes a robust hyperpolarizing shift (25 mV) in the voltagedependence of activation.24 As a consequence Nav1.7 channels open at more hyperpolarized potentials rendering dorsal root ganglion (DRG) neurons a hyperexcitable character, causing pain. However, the exact location of the activation gate in Nav channels remains unresolved. To test the hypothesis that the pore gate is located intracellularly, Chanda and colleagues have recently provided a model for pore-gating in Nav channels based on accessibility data derived from treatment of a Nav1.4 mutant channel with the thiol-modifying reagent MTSET together with analysis of the closed and putatively open-state crystal structures of bacterial sodium channels. They suggest that an intracellular gate composed of a ring of four hydrophobic residues, one from each S6 helix, which is highly conserved within the VGIC superfamily, is responsible for regulating access to the extracellular pore of Nav channels.25

Nav channel sensitivity to protons. Protons impart subtype-specific modulation of inactivation in neuronal, skeletal muscle, and cardiac Nav channels. Working with two pore mutant constructs of the human cardiac Nav1.5 channel, Jones and coworkers identify P-loops amino acids C373 and H880 as possible Nav1.5 proton sensors. The protonation of C373 and H880 may affect channel function and contribute to cardiac arrhythmia.26 Although the exact amino acid residues responsible for the changes in human Nav kinetics during the extracellular acidosis that occurs during cardiac ischemia are still unknown, studies using naked mole rats, 9 ACS Paragon Plus Environment


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which will be discussed in the pain section of this review, provide us with some insight about their existence and location.

Molecular architecture of β subunit. As mentioned in the introduction of this review, eukaryotic Nav channels are composed of one α subunit, which can be coupled to one or two β subunits. There are four genes in the mammalian genome (SCN1B–SCN4B) that encode five β subunit proteins designated β1, β1B, β2, β3, and β4. Subunits β2 or β4 bind to the α subunit via a disulfide bond whereas β1 and β3 subunits are associated non-covalently. All β subunits are transmembrane proteins, except β1B which is expressed as a soluble molecule. Although the α subunit alone is sufficient to form a fully functional Nav channel, β subunits play crucial roles in the fine tuning of channel kinetics and channel expression on the cell surface. Nav β subunits are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs) possessing an extracellular Ig domain that participates in a number of cell adhesion–related activities.27 Interestingly, studying the effects of β3 subunits on Nav channel structure and function, Jackson and coworkers found a more complex picture than expected. The X-ray crystal structure of a β3 subunit shows that they can trimerize via their Ig domains. Also on the surface of HEK293 cells, full-length β3 subunits predominantly adopt a trimeric structure. In addition, those studies showed that the Nav1.5 α subunit can bind β3 subunits at four different sites, suggesting the potential for formation of α subunit oligomers in vivo.28 It is noteworthy to mention that despite most of the current research focuses on modulating Nav channels using agents that bind to the α subunit, interaction with β subunits provides an indirect strategy for treating Nav-dependent pathologies.29 Raman and Lewis recently published several studies directed at understanding the function of the β4 subunit as an endogenous open-channel blocking protein. Using human Nav1.4


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channels expressed in HEK293T cells, they found that the β4 peptide competed with the fast inactivation process.30

GENERAL PHYSIOLOGY Nav channels are expressed in the cell membranes of excitable and non-excitable cells. The activation of Nav channels is voltage driven and the cellular membrane potential detected by the protein’s voltage sensor determines the state in which the channels reside. At hyperpolarized resting potentials the probability of Nav channels being in an open state is low. Membrane depolarization facilitates a conformational change of the α subunit induced by movements within the voltage-sensing domains, resulting in the opening of the Na+-selective channel pore. Nav channels open very quickly and mediate the inward current of Na+ underlying the rapid upstroke of the neuronal and cardiac action potentials in nerve and muscle fibers. Within milliseconds, open channels transition into a non-conducting inactivated state. Fast Nav channel inactivation refers to the rapid decay of sodium current (INa) observed in response to short depolarizations mediated by the closing of the intracellular inactivation gate. In contrast, slow channel inactivation develops when nerve and muscle fibers are depolarized for periods of seconds; this is caused by long-term changes in the resting membrane potential or during extended periods of repetitive neuronal firing.13 In general, Nav channels are kinetically fast transient channels that inactivate/close within milliseconds. However, under certain circumstances incomplete fast inactivation can generate persistent sodium currents. Subtype Nav1.9 is known to give rise to low-threshold persistent INa in sensory neurons. Also Nav1.4 and Nav1.6 channels have been reported to generate persistent currents in muscle fibers and Purkinje neurons, respectively. 11 ACS Paragon Plus Environment


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Mutations in SCN1A, SCN2A, SCN3A, and SCN8A cause defects in inactivation gating, increasing persistent INa leading to ataxia and epilepsy.31 In some neurons Nav channels open upon recovery from fast inactivation during membrane repolarization. This transient opening of the channel pore generates a large inward sodium current called resurgent current. It has been suggested that the resurgent currents are mediated by the relief of open-channel block by auxiliary β subunits. This phenomenon has recently received more attention in the pain field, since it has been shown that Nav1.7 channel mutations associated with pain disorders increase resurgent currents in dorsal root ganglia (DRG) neurons32 and that resurgent currents can be enhanced by inflammatory mediators such as bradykinin, histamine, PGE2, and ATP.33 Mutations of highly conserved, positively charged amino acids in the S4 segments of the voltage-sensing domain, can create a fast-activating and non-inactivating nonselective permeation pathway for Na+ and K+, designated the gating pore. Nav1.4 and Nav1.5 channel dysfunction due to gating pore currents, also called omega currents, was identified as the underlying mechanism of neuromuscular disorders in humans (vide infra).34 Nav channels are also expressed in cell types not considered excitable, such as astrocytes, microglia, macrophages, and cancer cells, and have been implicated in the regulation of phagocytosis, cell motility, and metastatic activity. Such noncanonical roles of Nav channels have recently been reviewed in detail.35

Genetic target validation through channelopathies. Mutations in the genes encoding the Nav α subunits and the β subunits can affect functional channel expression or alter gating properties of these channels. As a consequence mutations can lead to channel dysfunctions giving rise to abnormal neuronal firing and associated disease phenotypes called channelopathies. Nav channels have long been recognized as effective targets for treating neuronal and cardiac disorders. The development of transgenic preclinical models for individual 12 ACS Paragon Plus Environment

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Nav subtypes has led to a better understanding of the role of Nav channels in physiology and disease. Most important are recent reports that loss-of-function mutations in Nav1.7 are associated with congenital indifference to pain, and that Nav1.7 gain-of-function mutations are the underlying cause for hereditary human pain disorders. As a result the attention of many pharmaceutical and biotech companies, as well as academics, have been directed to Nav as a potential drug target for the treatment of pain. Furthermore, recent genetic studies have expanded our knowledge of the role of sodium channels in diseases such as muscle and immune system disorders, migraines, epilepsy, multiple sclerosis, diabetes, cough, autism, and cancer, opening up a plethora of potential, novel pharmacological treatment options (Figure 2).36

THERAPEUTIC APPLICATIONS Pain. Primary sensory neurons with peripheral axons terminating in the skin or viscera and central axons terminating in the spinal cord, are responsible for detecting noxious stimuli perceived as pain. Specialized ion channels and receptors in peripheral terminals are involved in facilitating a generator potential that can be transformed into an action potential leading to neurotransmitter release at central projections, activation of second-order sensory neurons, and the transport of pain signals to the brain. Subtypes Nav1.3, Nav1.7, Nav1.8, and Nav1.9 have been identified as key players in nociceptive signaling. Nav1.7 channels are preferentially expressed in peripheral terminals of sensory neurons, within the neuronal soma of DRGs, and at central terminals of neuronal afferents in the superficial laminae of the spinal cord (Figure 2). They can also be found in trigeminal and sympathetic ganglion neurons, visceral sensory neurons, and olfactory sensory neurons. Nav1.7 13 ACS Paragon Plus Environment


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channels activate and inactivate rapidly but recover slowly from inactivation. Nav1.7 is considered a threshold channel producing substantial ramp currents in response to small depolarizations thereby enabling neuronal firing triggered by subthreshold stimuli. Nav1.7 channels have also been reported to generate resurgent currents in DRG neurons. Genetic studies on patients with hereditary pain disorders such as erythromelalgia (IEM), paroxysmal extreme pain disorder (PEPD), and small fiber neuralgia (SFN), revealed missense mutations in SCN9A, the gene encoding Nav1.7. Subsequent biophysical studies demonstrated that these mutations were in fact gain-of-function mutations that modulate Nav channel activation and/or inactivation properties leading to hyperexcitability in DRG neurons.37 Nav1.7 variants have also been shown to play a role in promoting axon degeneration38 and more recently in paroxysmal itch.39 In contrast, loss-of-function mutations of Nav1.7 leading to the expression of nonfunctional channel proteins have been found in families with congenital insensitivity to pain.40 Affected individuals feel no pain during childbirth, tooth extractions, or on suffering bone fractures or severe burns, clearly indicating a key role of Nav1.7 in pain signaling. Their cognitive and motor development is normal and no other anomalies in the PNS, such as sense of touch and temperature discrimination at the CNS, can be observed. Interestingly, Nav1.7 knockout mice do not feed and newborns die from starvation presumably due to anosmia.41 This is suggested by the fact that Nav1.7 is the predominant Nav channel in rodent olfactory sensory neurons.42 The deletion of Nav1.7 allows the generation of odor-evoked action potentials by olfactory sensory neurons; however, the neurons fail to initiate synaptic signaling from axon terminals at the first synapse in the olfactory system.43 Correspondingly, humans with homozygous SCN9A-null mutations have been diagnosed with congenital anosmia, an inability to smell that provides evidence of the expression of Nav1.7 channels in olfactory sensory neurons. Recently, researchers at Amgen


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were able to develop genetic and animal husbandry strategies that made it possible to circumvent lethal aspects of this phenotype and raise global Nav1.7 knockout mice. Notably, knockouts were anatomically normal, reached adulthood, and displayed a phenotype analogous to human congenital indifference to pain.44 The role of Nav1.7 in sympathetic neurons is not clear. Patients with complete insensitivity to pain do not report sympathetic deficits.40 Interestingly, it was observed that knocking out SCN9A in DRG neurons alone does not cause a total loss of pain in rodents, whereas ablation in both sensory and sympathetic neurons recapitulates features of complete human insensitivity to pain.45 More information is needed to determine the role of sympathetic Nav1.7 channels in pain signaling. Acid is a noxious stimulus that causes pain in vertebrates with one interesting exception, the naked African mole rat. Naked mole rats live in large underground colonies in which high concentrations of CO2 can be generated. High CO2 concentrations can cause tissue acidification and acid-induced pain. However, naked mole rats have adapted to their habitat in a peculiar way: they possess a tripeptide sequence in the S5–S6 linker of DIV in their Nav1.7 α subunit. This enhances proton-induced blockade of these channels, thereby preventing action potential firing in the rat’s nociceptors and consequently reduced acid nociception. This unique adaptive mechanism offers insight into inflammatory pain processes in which tissue acidosis plays a role.46 In addition to Nav1.7 channels, TTX-resistant Nav1.8 is expressed in DRG neurons, and in trigeminal and nodose ganglion neurons. Nav1.8 channels activate at more depolarized membrane potentials than Nav1.7 and mediate most of the inward sodium currents during the depolarization phase of neuronal action potentials that are critical for transmission of action potentials and repetitive firing. Heterozygous gain-of-function mutations in SCN10A, the gene 15 ACS Paragon Plus Environment


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encoding Nav1.8, cause hyperexcitability and aberrant firing of DRG neurons and have been linked to painful small-fiber neuropathies in humans, a finding that suggests an important role for Nav1.8 in nociceptive neuron function.47 Furthermore, rodent knockout and knockdown studies suggest the involvement of Nav1.8 in inflammatory and neuropathic pain.48 Nav1.8 could also be part of the mechanism linking metabolic disease and neuropathic pain in diabetic patients. Elevated glucose levels during hyperglycemia have been shown to cause increased formation of the highly reactive dicarbonyl metabolite methylglyoxal. It is believed that methylglyoxal depolarizes sensory neurons and induces post-translational modifications of Nav1.8 channels so that electrical excitability and firing of nociceptive neurons increase. In the clinic, plasma levels of methylglyoxal above 600 nM discriminate between diabetic patients with or without pain.49 Evolutionary adaption of the grasshopper mouse to its habitat, the desert of Arizona, provides remarkable evidence for the role of Nav1.8 in nociception. Peptide toxins isolated from scorpion venoms have been shown to activate neuronal Nav channels or impair channel inactivation, thereby increasing neuron excitability causing pain and/or paralysis. Interestingly, the grasshopper mouse (Onychomys torridus) kills and consumes bark scorpions while showing little signs of discomfort when stung by the scorpion. Biophysical studies showed that, due to amino acid variants in the Nav1.8 channel of the grasshopper mouse compared to the regular mouse (Mus musculus), the scorpion venom blocks the channels, thereby inhibiting the transmission of pain signals into the CNS. In contrast, Nav1.8 channels expressed in nociceptive neurons of regular mice were not inhibited by the venom, so that these animals perceived intense pain.50


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A non-inactivating INa component found in DRG neurons and trigeminal ganglion neurons is mediated by Nav1.9 channels, encoded by the gene SCN11A, a Nav channel subtype that activates at highly hyperpolarized membrane potentials close to the resting potential. Nav1.9 channels do not contribute to the action potential upstroke. Instead, these channels help control the membrane potential thereby regulating neuronal excitability. Recently, specific heterozygous mutations in SCN11A have been linked to congenital inability to feel pain in humans.51 In contrast to individuals with SCN9A mutations, the sense of smell is intact in individuals carrying SCN11A mutations. However, SCN11A mutations have been associated with mild muscular weakness. Heterozygous knock-in mice carrying the ortholog mutations also showed reduced sensitivity to pain, reproducing in part the human phenotype.51 Genome-wide linkage studies in Chinese families with episodic pain disorders have revealed several gain-of-function Nav1.9 mutations.52 Like Nav1.7 and Nav1.8 gain-of-function mutations, Nav1.9 mutations modulate voltage-dependent channel gating properties, resulting in depolarized membrane potentials, likely leading to an increased excitability of DRG neurons.53 To date no clear monogenic diseases associated with mutations in SCN3A, the gene encoding Nav1.3 have been identified. However, rodent studies that demonstrate increased Nav1.3 transcript levels in sensory neurons following axotomy and neuronal inflammation suggest Nav1.3 channels are potential contributors to neuronal hyperexcitability and pain.54 Most Nav blockers for pain treatment on today’s market are local anesthetics (LA), class I cardiac antiarrhythmics (e.g., lidocaine, mexiletine), anticonvulsants (e.g., carbamazepine), and antidepressants (e.g., amitriptyline).1b Antidepressants and anticonvulsants show good CNS penetration, thereby inhibiting Navs not only in the periphery, but also in the brain and spinal cord. These drugs are nonselective Nav blockers (Figure 5A), displaying state- and frequency17 ACS Paragon Plus Environment


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dependence of block, and share a common binding site within the pore of the Nav α subunit (LA site, Figure 4). Although clinically effective, due to their global Nav activity, the full analgesic potential of these agents is limited by numerous adverse events related to the CNS and cardiovascular systems, such as dizziness, sedation, convulsions, and cardiotoxicity. For example, carbamazepine and mexiletine show efficacy in the context of specific Nav1.7 gain-offunction mutations causing pain disorders but the therapeutic window is limited. To avoid the side effects occurring with systemic administration, topical lidocaine patches are applied to affected areas. A recent small study in humans suffering from inherited erythromelalgia demonstrated that the novel, potent, but nonselective Nav1.7 channel blocker XEN-402 (structure not disclosed, Nav1.7 IC50 = 80 nM) significantly reduced the amount of pain experienced by patients, a finding that offers hope for discovering potent and preferentially selective Nav1.7 inhibitors for pain therapy.55 In 2013, McCormack and coworkers showed that subtype-selective small molecules 16 and 17 (ICA-121431 and PF-04856264, Figure 9) target a novel binding site within the Nav voltage sensor of DIV (ICA/PF site, Figure 4).56 These molecules are potent Nav1.1, Nav1.3, Nav1.6, and Nav1.7 inhibitors and show remarkable selectivity over cardiac Nav1.5 channels (~ 1000-fold). The study demonstrated the feasibility of subtype-selective Nav channel inhibition with small molecules by targeting interaction sites away from the conserved pore region.56 XEN-402, 16 and 17 will be further discussed in the clinical update section of this review. Studies on natural neurotoxins isolated from the venoms of spiders, snakes, scorpions, sea anemones, and other animals have shown that Nav channels can be selectively inhibited or their function can be selectively modulated by targeting other sites on the α subunit that are less conserved than the pore-forming region.57 µ-SLPTX-Ssm6a is a highly Nav1.7-selective peptide 18 ACS Paragon Plus Environment

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toxin isolated from the venom of a centipede; it will be discussed in the peptidic natural product section of this review. µ-SLPTX-Ssm6a inhibits Nav1.7 by shifting of the voltage-dependent activation to more depolarized potentials, a hallmark of the channel gating modification induced by other Nav peptide toxins such as Protoxin II (Figure 3E).58 Although exactly where interaction of µ-SLPTX-Ssm6a occurs on the α subunit is currently unknown, its proposed mechanism of action involves binding to the voltage sensor of the channel. µ-SLPTX-Ssm6a appeared to be a more potent analgesic than morphine in the studies conducted on chemical-, thermal-, and acidinduced pain in rodents.59 However, the group did not report on the inhibitory effects of the synthetic version of the peptide. Most recently, the generation of a highly selective and potent inhibitory monoclonal antibody against Nav1.7 was reported.60 The antibody SVmab1 was raised against the S3–S4 linker of the voltage-sensing domain DII and prevents channel activation by stabilizing the channels in the closed state thus shifting the voltage dependence of activation to more depolarized voltages. The antibody displays exquisite selectivity for Nav1.7 (IC50 = 31 nM), showing little activity on Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, and Nav1.8 (IC50s in the µM range) in patch clamp studies. Like small molecules 16 and 17, and presumably µSLPTX-Ssm6a, SVmab1 inhibits the channel by binding to a channel region away from the pore, effectively suppressing inflammatory and neuropathic pain after intravenous, intrathecal, or intraplantar administration in mice. Furthermore, spinal cord slice recordings in the presence of SVmab1 revealed the suppression of nociceptive transmission in spinal cord dorsal horn neurons, thus confirming a role for Nav1.7 in spinal cord nociceptive transmission. Interestingly, the antibody also inhibited acute and chronic itch in preclinical mouse models, suggesting Nav1.7 channels as potential targets for itch management. 19 ACS Paragon Plus Environment


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As previously discussed, selective inhibition of Nav1.7 channels in sensory neurons of the PNS appears to be a promising approach to pain pharmacotherapy. Furthermore, data recently generated with Nav1.7-specific monoclonal antibody SVmab1 administered systemically and intrathecally, suggests that Nav1.7 controls pain not only in the periphery but also via a central mechanism.60 We previously noted that the clinical phenotype of Nav1.7-related congenital insensitivity to pain is accompanied by anosmia, a side effect that could be manageable should pharmacological Nav1.7 inhibition result in loss of smell. On the other hand, insensitivity to pain induced by Nav1.9 mutations has been associated with gastrointestinal motility disturbances and muscle weakness, a potential clinical problem should pharmacological modulation of Nav1.9 reproduce symptoms of the genetic phenotype.61 Additionally, Nav1.8 and Nav1.9 channels have been previously detected in retinal amacrine and ganglion cells, photoreceptors, and Muller glia, raising concerns that drugs targeting these channels could alter visual processing in the retina.62 In summary, strong genetic validation and the potential for predictable and manageable side effects makes Nav1.7 an irresistible target for researchers in the pain field. As will be discussed in the clinical update section of this review, Convergence has recently demonstrated pharmacological proof of concept in two phase II pain trials.

Epilepsy. Nav1.1 and Nav1.2 channels are preferentially expressed in GABAergic interneurons that synthesize and release GABA, the major inhibitory neurotransmitter in the brain. Interneurons are crucial elements in the regulation of neuronal network excitability and synchronization of neuronal activity. Missense and loss-of-function mutations in Nav1.1 and Nav1.2 impair the excitability of GABAergic inhibitory neurons and create neuronal hyperexcitability and several forms of epilepsy. More than 20 Nav1.1 mutations have been 20 ACS Paragon Plus Environment

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implicated in the autosomal dominant epilepsy disorder GEFS+ (generalized epilepsy with febrile seizures plus). More than 600 Nav1.1 mutations have been found to be associated with Dravet syndrome, a severe myoclonic epilepsy of infancy. Compared to mutations in Nav1.1, the consequences of mutations in Nav1.2 are less severe; they can lead to benign familial neonatalinfantile seizures, a mild seizure syndrome that usually responds favorably to antiepileptic drugs and remits by one year of age.36h Anticonvulsants, such as the Nav blockers phenytoin, carbamazepine, lamotrigine, and valproate are effective in suppressing the abnormal neuronal firing underlying epileptic seizures. These drugs are state- and frequency dependent non-selective, Nav inhibitors, preferentially binding and stabilizing channels in the non-conducting inactivated state.36h In contrast, it has been proposed that lacosamide, a more recently approved anticonvulsant drug for the treatment of epilepsy, achieves its therapeutic effects by enhancing slow inactivation of neuronal Nav channels.63 Additional clinical trials have been performed to explore lacosamide’s efficacy in painful diabetic neuropathies. Recent mechanistic studies have suggested interactions of lacosamide with closed Nav channel states preceding the open state.64 Nav1.1 channels are the most important sodium channels for action potential initiation in the fast-spiking subtype of GABAergic interneurons, so selective activation of Nav1.1 channels presents a potential new treatment approach for epilepsy. As we will see in the clinical update section of this review, this approach is complemented by Xenon’s ongoing efforts targeting Nav1.6 for the potential treatment of Dravet syndrome.

Neuromuscular disorders. Nav1.4 channels are essential for the generation and propagation of action potentials that trigger muscle contraction. Mutations located throughout the α subunit, including both the voltage-sensing domain and the pore domain, can increase channel 21 ACS Paragon Plus Environment


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activity and have been identified as the underlying cause of four hereditary sodium channelopathies, hyper- and hypokalaemic periodic paralysis, paramyotonia congenital, and congenital myasthenic syndrome.36a The typical symptoms of the diseases are myotonia and muscle weakness caused by uncontrolled repetitive muscle fiber discharges. Interestingly, detailed studies of the biophysical changes induced by mutations causing hypokalaemic periodic paralysis revealed omega currents through the voltage-sensing domain of the channel.65 Although a number of agents have been identified that interact with the voltage-sensing domain by acting as channel gating modifiers, to date no gating pore blockers have been described other than divalent cations, such as Ba2+, Zn2+, and Ca2+ (mM concentration).34b However, compounds that would block the gating pore of Nav1.4 without impinging on the critical movements of the voltage-sensing domain may offer novel approaches for the treatment of some neuromuscular disorders. Meanwhile, local anesthetics and antiarrhythmics such as mexiletine and flecainide are the only available treatment for some of the skeletal muscle sodium channelopathies, and their effects are variable.

Cardiovascular diseases. Nav1.5 channels mediate the rising phase of the cardiac action potential. Many gain-of-function mutations in the gene encoding Nav1.5, SCN5A, have been described to date. Patients carrying such mutations show signs of cardiac dysfunction diagnosed as long QT syndrome. The majority of these mutations disrupt fast sodium channel inactivation, impairing the ability of the channel to close completely and thereby generating persistent sodium currents and a prolongation of the ventricular action potential.66 In contrast, loss-of-function mutations of SCN5A produce truncated, nonfunctional channel protein or impair the membrane trafficking of Nav1.5. Altered cardiac action potentials lead to ventricular arrhythmias and in extreme cases to Brugada syndrome, in which fibrillation may lead to death.66 22 ACS Paragon Plus Environment

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Interestingly, recent genetic studies have also implicated SCN10A variants (Nav1.8) with disturbances in cardiac conduction and Brugada syndrome, demonstrating the emergence of SCN10A as a cardiac arrhythmia susceptibility gene.67 Class I antiarrhythmic drugs, such as lidocaine, mexiletine, flecainide, quinidine and procainamide, play an important clinical role in the therapy of cardiac arrhythmias. These drugs act as frequency-dependent inhibitors of cardiac sodium channels. Drug induced channel block accumulates with increasing firing frequencies during tachyarrhythmias, thereby slowing the rate of action potential generation in the cardiac myocyte and dampening excitability in the heart.66 Paradoxically, these same drugs and other nonselective Nav blockers have the potential to induce cardiac arrhythmias in normal healthy individuals. In summary, the risks of cardiac Nav1.5 channel modulation are clearly highlighted by the clinical Nav1.5 genetics associated with cardiac dysfunction and by adverse cardiac effects caused by off-target Nav1.5 activity of pharmacological reagents. Therefore, unless the goal is development of novel antiarrhythmics, activity on Nav1.5 channels must be avoided when trying to identify new selective Nav inhibitors for other indications.

Respiratory disorders. Cough is a symptom of a number of diseases that affect the respiratory system, and the use of Nav inhibitors to treat cough has emerged as a very promising therapy. The cough reflex in mammalian airways is regulated by vagal sensory neurons expressing Nav channels. Local anesthetics are effective antitussive agents in humans and animals when applied locally, presumably by inhibiting action potentials of airway afferent neurons. However, the effect is short-lasting and of little use for the treatment of pathological cough. Studies in the guinea pig have shown that the vagal cough receptors and neurons innervating the respiratory tract express Nav1.7, Nav1.8, and Nav1.9 channels. Nav1.7 is particularly highly expressed in vagal ganglia. Using the gene silencing technique (small hairpin 23 ACS Paragon Plus Environment


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RNA) to knock down Nav1.7 expression in the vagal sensory ganglia of guinea pigs abolished action potential conduction and significantly suppressed coughing induced by citric acid.36c,68 Hence the selective pharmacological inhibition of peripheral Nav1.7 channels could have antitussive effects. Importantly, selective inhibition of Nav1.7 without affecting other TTXsensitive channels will be critical because systemic administration of tetrodotoxin through food poisoning by consumption of contaminated fugu fish inhibits all TTX-sensitive channels leading to rapid death from respiratory cessation due to paralysis of the diaphragm.69

Cancer. Ion channels such as Navs play an important control function in a range of cellular processes involved in metastasis and angiogenesis. Nav channel function and expression are regulated by hormones, growth factors, cytokines, and hypoxia, and the channels control cancer cell invasiveness by regulating cell motility and the secretion of proteolytic enzymes. The pathological upregulation of Nav channels can make cancer cells highly invasive. In fact, evidence exists that the levels of Nav expression are correlated with invasiveness and metastatic potential in several types of cancer. For example, the overexpression of Nav1.5 channels in cancer cells has been linked to strong metastatic potential.35,70 However, upregulation of several Nav subtypes and β subunits, has been associated with different cancers, such as metastatic carcinomas of prostate, breast, small cell lung cancer, neuroblastoma, melanoma, cervical, ovarian, and colon cancer.70,71 The nonselective Nav inhibitors ranolazine, riluzole, lidocaine, phenytoin, and carbamazepine have been shown to block metastatic cell behavior for in vitro models,72 suggesting a potential for cancer therapy; however, the validity of Nav channel inhibition as a therapeutic approach to curb invasiveness and metastatic potential of cancerous tumors is still questionable. Another therapeutic avenue to be explored is the diagnostic use of Nav channel expression patterns observed in tumor tissues as cancer biomarkers.73 24 ACS Paragon Plus Environment

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Other potential therapeutic applications. A number of research articles have revealed a potential role for Nav channels in treating a variety of neuronal medical conditions, such as diabetic neuropathy, cognitive dysfunction, autism, and multiple sclerosis, where there may be opportunities for developing novel treatments. Diabetes mellitus is commonly associated with painful peripheral neuropathy and it has traditionally been thought that pathological changes during diabetes may be the cause of neuropathy. Recently Hoeijmakers and coworkers came up with the intriguing hypothesis that painful neuropathy is not a complication of diabetes but rather occurs as a result of Nav1.7 mutations conferring vulnerability to injury in pancreatic β cells and DRG neurons. It has been suggested that dysfunction of Nav1.7 expressed in pancreatic β cells and DRGs increases susceptibility to development of diabetes via β cell injury and, via a different mechanism, of painful neuropathy.36i Impaired cognitive performance associated with reduced expression of Nav1.1 in heterozygous SCN1A +/– mice, together with reduced short-term memory observed in otherwise healthy humans carrying SCN1A mutations, suggest a potential link between reduced Nav1.1 expression and cognitive deficits. As a consequence Nav1.1 channel activators were proposed for the potential symptomatic treatment of cognitive dysfunctions associated with Alzheimer’s disease and schizophrenia.74 In addition, recent studies reported strong links between two independent Nav1.2 nonsense mutations, splice site mutations, and autism,36b,36e,36f while Nav1.1 mutations were shown to be associated with hemiplegic migraine type 3.75 The function of Nav subtypes Nav1.2, Nav1.6, and Nav1.8 appear to play a role in the pathophysiology of multiple sclerosis (MS), a disease characterized by axonal demyelination and degeneration, and consequent cerebellar dysfunction, presumably due to dysregulated immune responses. Thus, Nav1.2 and Nav1.6 channels have been implicated in the restoration to neuronal 25 ACS Paragon Plus Environment


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conduction after demyelination and axonal degeneration. Furthermore, the upregulation of Nav1.8 channels observed in an experimental mouse model of MS (EAE; experimental autoimmune encephalomyelitis) may contribute to cerebellar dysfunction. Consistently, MS-like deficits observed in the mouse model could be partially reversed with the Nav1.8 blocker A803467.76 Also, nonselective Nav blockers phenytoin and flecainide have been demonstrated to be axon-protective in experimental rodent models of multiple sclerosis. Therefore, pharmacological modulation of Nav channel function and/or manipulation of channel expression in neuronal membranes of MS patients may be helpful in modifying disease progression.77 In summary, we have illustrated the important role of Nav channels in physiological processes and the consequences of Nav channel dysfunction, often caused by mutations, leading to a broad spectrum of diseases associated with various genetic phenotypes. Because of their key role in neuronal conduction and muscle contraction, Nav channels have been successfully targeted for decades by antiepileptic and antiarrhythmic agents, and by local anesthetics. However, currently available Nav channel modulators are not selective and often suffer from a limited therapeutic index. Genetic studies in recent years have pinpointed the role of specific Nav channel subtypes in certain disease phenotypes and hence identified novel, genetically validated drug targets. The Nav arena holds promise, and research investment from academics, biotech and pharmaceutical industry is substantial. The key to success for any novel therapeutic Nav channel modulators will be increased potency and selectivity that can improve efficacy and safety margins relative to existing nonselective treatment options.

NATURAL PRODUCTS AND THEIR DERIVATIVES 26 ACS Paragon Plus Environment

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Throughout history, envenoming by animal and plant toxins has fascinated humans. By producing toxins that target Nav channels, venomous animals and poisonous plants exploit the excitatory and inhibitory action of toxins to incapacitate prey or defend against predators (Figure 3). Not only do toxins from scorpions, sea anemones, spiders, cone snails, insects, and plant extracts serve as important chemical tools for understanding Nav channel structure, pharmacology and physiology, but they also provide extraordinary leads for developing novel subtype-selective agents that have therapeutic potential.

Non-peptidic natural products. Tetrodotoxin, saxitoxin and zetekitoxin. Tetrodotoxin (TTX, 1, Figures 3A and 6)78 and saxitoxin (STX, 2, Figure 6)79 are neurotoxins whose biology and chemistry have been extensively studied. They serve as an important reminder of the vital role of natural products in drug discovery research. TTX and STX are secondary guanidinium metabolites that block Nav channels and have estimated lethal doses of 0.5 mg for a 75-kg person. TTX inhibits Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, and Nav1.7 channels with IC50 values in the single nanomolar range. TTX has a rigid structure comprised of a 10-atom carbon–oxygen cage and a fused 6-membered guanidinium ring. It is present mainly in marine species such as puffer fish, blue ringed octopus, and some worms, shellfish, and crustaceans, although it can also be found in terrestrial animals such as some amphibians and in bacteria. Whether TTX accumulates after dietary administration or it is biosynthesized in each of the above species still remains to be clarified. On the other hand, it is well-known that STX is produced by single-celled dinoflagellate algae and freshwater cyanobacteria, although it is most commonly associated with oceanic red tides and shellfish poisoning. STX is an unusual tricyclic bisguanidinium compound with functional activity equivalent to that of TTX. When occupied, the guanidinium toxic binding site resembles a conical hole plugged by a stopper. In this site, 27 ACS Paragon Plus Environment


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named Site 1 (Figure 4), negatively charged carboxylate groups of acidic amino acid residues form two charged rings at the outer pore of the Nav channel. These interact with the positively charged guanidinium and hydroxyl groups of TTX and STX. However, complete details of how TTX and STX bind to eukaryotic Nav channels is still unknown. Chen and Chung examined the inhibition the Nav1.4 subtype by TTX in atomic detail using MDS and corroborated the presence of a H-bond network between the guanidinium moiety of TTX and the four carboxylic acid side chains that form the outer ring. Interestingly, it was observed that the guanidinium group of TTX adopts a lateral orientation, as opposed to protruding into the SF as previously thought.80 Among the isolated natural products analogous to TTX and STX, the TTX metabolite 4 (4,9anhydro-TTX, Figure 6) and zetekitoxin AB (ZTX, 3, Figure 6) are especially interesting. Despite its close structural similarity to TTX, metabolite 4 is a Nav1.6-selective inhibitor, being 161-, 44-, 127-, and 163-fold selective at the mammalian Nav1.2, Nav1.3, Nav1.4, and Nav1.7 channel subtypes, respectively, that were expressed in Xenopus laevis oocytes.81 ZTX (3, Figure 6) is a very structurally complex toxin isolated from the skin of a Panamanian golden frog. ZTX is an extremely potent Nav inhibitor with IC50 values of 6.1, 62, and 280 pm against Nav1.2, Nav1.4, and Nav1.5, respectively.82 A number of deoxy-TTX derivatives have been isolated or synthesized, and in general they have much lower inhibitory activity than TTX. One exception is the recently isolated deoxy-analogue 5 (6-deoxy-TTX, Figure 6), whose EC50 was found to be three times greater than that of TTX in a mouse neuroblastoma cell–based assay.83 Working on the development of a subtype-selective TTX/STX-like compound, Sakate, Y. et. al have recently synthesized 6 and 7 (8-deoxy-TTX and 5-deoxy-TTX, respectively, Figure 6).84 Analogue 6 retained significant inhibitory activity in a cell-based assay, suggesting that the C-8 position of TTX could be modified to produce subtype-selective blockers of Nav channels.84 28 ACS Paragon Plus Environment

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Ciguatoxins and brevetoxins. Ciguatoxins (CTXs, such as CTX-1, 10, Figure 7) and brevetoxins (PbTxs, such as PbTx-2 and PbTx-3, 8 and 9, Figure 7)85 are non-peptidic ladderframe polyether toxins produced by marine microalgae dinoflagellates, which grow in tropical and subtropical regions, mainly of the Indo-Pacific Oceans and the Caribbean Sea. CTXs are found on dead coral surfaces and bottom-dwelling algae, and PbTxs are responsible for the red tides off the Florida coast and Gulf of Mexico that cause massive fish kills and mortalities in birds, marine mammals, and other marine species. These toxins accumulate in tissues of fish that eat the algae and bioaccumulate through the food chain where they are oxidized to more toxic metabolites before being ingested by humans. These marine toxins interact with Site 5 of Nav channels located on DIV of the α subunit (Figure 4). The common pharmacophore of these toxins consists of a cigar-shaped molecule approximately 30 Å long that binds to Site 5, primarily through hydrophobic interactions and strategically located hydrogen-bond donors.86a Instead of being inhibitors like TTX and STX, these toxins are activators of various Nav channel subtypes and increase Na+ permeability in nerve cells. Interestingly, the Nav1.8 subtype found in the PNS seems particularly susceptible to ciguatoxins,86b causing a large persistent current upon toxin exposure that seems to be responsible for the burning pain and electric shock–like sensations experienced. Type B brevetoxin PbTx-3 show tissue selectivity in humans and rats, binding to the heart tissue with a significantly lower affinity than to skeletal muscle and brain tissues.87 Ciguatera fish poisoning (CFP) is caused by consuming ciguateric reef fish. The initial symptoms consist of gastrointestinal effects—nausea, diarrhea, and abdominal pain—and usually disappear within 2 days to 1 week. The neurological signs, which develop after 2–5 days and can last for months or even 1 year, include paresthesias, painful dysesthesias, ataxia, and cold 29 ACS Paragon Plus Environment


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allodynia. Trying to understand how brain neuron and astrocyte signals underlie the mechanism of ciguatera poisoning, Li and coworkers isolated CTX-1 from the viscera of moray eels and administered it orally or cerebroventricularly to rats. They showed that CTX-1 elicited neuronal hyperexcitability, facilitating synaptic transmission and blocking the induction of long-term potentiation in the anterior cingulate cortex.88 Due to globalization, ciguatoxin-containing fish can be consumed anywhere in the world, and CFP is currently a growing social problem. Between 50,000 and 200,000 cases of CFP are reported each year and these numbers are expected to grow because of global warming,89 so a major effort is currently underway to develop better detection and quantification technologies, and methods to attenuate the neurotoxic effect of these toxins.90

Batrachotoxin and veratridine. Batrachotoxin (BTX, 11, Figures 3B and 8) is a complex steroidal alkaloid isolated from the skin secretion of frogs of the genus Phyllobates found in South and Central America.91 BTX is a selective agonist that causes a complex array of responses at Navs; these include hyperpolarization of threshold activation, elimination of inactivation gating, and reduction of single-channel conductance causing in vivo locomotor difficulties, partial paralysis, convulsions, and then death (15–20 min in mice, LD50 = 2 µg/kg). Homology modeling and protein mutagenesis data suggest that BTX binds to the inner pore region (Site 2, Figure 4) of Navs, where interactions with the C, D, and E rings of BTX are critical for its affinity. Embera Chocó Indians of western Colombia use extracts from dart frog skin to poison their blowpipe darts when hunting animals such as reptiles, birds, and mammals for food. One frog has enough poison to prepare around 50 hunting darts. Because Phyllobates frogs in captivity are entirely free of BTX, it is believed that this toxin has a dietary origin. This is supported by the fact that BTX is also found in the feathers, skin, and internal organs of 30 ACS Paragon Plus Environment

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Pitohui and Ifrita Papua New Guinea birds. The presence of BTX in these birds has been linked to the consumption of small beetles of the melyrid family.92a However, while identifying the specific diet of frogs containing more or less BTX, Mebs and coworkers have demonstrated that the ability of the frogs to sequester the diet alkaloids also plays a crucial role in determining BTX levels.92b BTX and its analogs are currently being explored for their potential to treat pain and other diseases of the nervous system. Unfortunately, SAR and pharmacology studies of BTX and their analogs have been limited by difficulties in obtaining BTX, either from natural sources or synthetically. To overcome these challenges, Du Bois and coworkers have recently developed a method of synthesizing a furan-derived C-D-E intermediate 12 (Figure 8) to provide access to BTX and A–B ring BTX derivatives.93 Veratridine (VTD, 13, Figure 8) is another steroidal alkaloid neurotoxin isolated from the seeds of the Central America and Mexican plant sabadilla lily.94a Although VTD shares the same binding site (Site 2) as BTX, VTD is an agonist at Navs and is less potent than BTX (LD50 = 9 mg/kg in mice). To characterize the interaction mechanisms of VTD with Navs, Yamagaki and coworkers have identified the hydrophobic VTD binding surface and a novel VTD binding residue Leu14 at DIVS6 using NMR titration experiments.94b

Other non-peptidic natural products. The endocannabinoid anandamide isolated from porcine brain and also found in human brains,95a and the catechin (–)-gallocatechin-3-gallate (GCG, 14, Figure 8)95b found in cocoa tea have analgesic properties that could be attributed to inhibition of Navs. Although much less is known about the activity of these natural products on Navs, Okura and coworkers have shown that anandamide inhibited Na+ currents in Nav1.2, Nav1.6, Nav1.7, and Nav1.8 α subunits expressed in Xenopus oocytes.96a Jiang and coworkers



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have recently shown that GCG potently inhibits tetrodotoxin-resistant Na+ currents in rat DRG neurons.96b Marine macrocyclic natural products form another class of non-peptidic Nav ligands. Although this class of compounds has not received much attention, antillatoxin (15, Figure 8),97 a natural product isolated from a marine cyanobacteria, is a potent Nav agonist that binds at Site 2. Exposing goldfish to antillatoxin in sea water revealed a highly ichthyotoxic potency (LD50 = 0.05 µg/mL),97a a toxicity exceeded only by brevetoxins.

Peptidic natural products. Intensive efforts by medicinal chemists have provided small molecules that modulate the activity of ion channels, but they often lack selectivity and/or potency, a “must have” in order to validate new therapeutic Nav channels. During millions of years of evolution, natural selection has favored animals whose venoms contain peptides (neurotoxins) that block or modify the function of ion channels. Millions of unique disulfide-rich peptides from venomous spiders, scorpions, and mollusks, some of them known, but the vast majority remaining undiscovered, serve as a tremendous, invaluable pool of novel agonists and antagonists with superb selectivity and potency against specific Nav subtypes. Proteomic and transcriptomic analyses have revealed that individual spider and cone snail venoms can comprise more than 1,000 distinct peptides, and scorpion venoms often contain as many as several hundred components. To facilitate the search for new therapeutic leads, several technologies have recently been developed to identify, isolate, and synthesize those peptides present in the enormous diversity of venom-derived neurotoxins. In this section we summarize recent findings in the major venomous species already mentioned, with emphasis on peptides that possess the highest potency and subtype-selectivity at mammalian Navs. 32 ACS Paragon Plus Environment

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Cone snail toxins. Cone snails, slow moving and unable to swim, are unique among the venomous species for their ability to deliver via a harpoon a diverse array of small disulfidebridged conopeptides or conotoxins for prey capture. At present only about 0.1% of conopeptides have been characterized pharmacologically. There are a number of cone snail venom peptide families that act on VGICs, ligand-gated ion channels, G protein-coupled receptors (GPCRs), and neurotransmitter transporters.98 Among these families four are known to target Nav channels, µ-, µO-, δ-, and ι-conotoxins, although a fifth family, µO§-conotoxins, has been recently identified.99 While µ-, µO-, and µO§-conotoxins block Nav channels, δ- and ιconotoxins activate them. Importantly, these families of conotoxins provide some of the most subtype-selective Nav modulators, especially for Nav1.2, Nav1.4, and Nav1.8. Among the conotoxins targeting Navs, µ-conotoxins are the most numerous and best characterized. They consist of 16 to 26 residues with six cysteine amino acids forming three conserved disulfide bonds that stabilize their three-dimensional structure. They possess a net positive charge (+2 to +7), which allows them to electrostatically bind to and block the negatively charged Nav channel pore or Site 1 (TTX/STX site). Although µ-conotoxins overlap the TTX binding site, µconotoxins interact with a larger number of Nav residues than TTX, resulting in a superior subtype-selectivity profile compared to TTX. Most µ-conotoxins have the highest affinity for the Nav1.4 subtype allowing cone snails to immobilize their pray. Recently, Zhang and coworkers have shown that co-expression of Nav β subunits alters the affinities of µ-conotoxins.100 Interestingly, TTX and STX affinities were not altered by Nav β subunit co-expression. The first µ-conotoxins discovered were GIIIA and GIIIC,101a which selectively target the skeletal muscle subtype Nav1.4. By stepwise replacement of the three cysteine residues with alanine in GIIIA, Sato and coworkers have demonstrated that all three 33 ACS Paragon Plus Environment


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disulfide bridges are essential for stabilizing the specific conformation of GIIIA needed for biological activity.101b Another important µ-conotoxin, KIIIA (Figure 3D) is a 16-amino-acid peptide that shows potent analgesic activity following systemic administration in mice.102 Despite being smaller than most of the peptidic toxins discussed in this section, KIIIA it is the most potent blocker of the Nav1.7 subtype (Kd = 0.29 µM) in a panel of 11 µ-conotoxins.103 KIIIA also potently inhibits Nav1.1 and Nav1.2 (Kd = 0.29 and 0.005 µM, respectively), as well as Nav1.4, and Nav1.6 (IC50 = 0.09 and 0.24 µM, respectively). Due to its small size, attractive selectivity profile, and analgesic efficacy in vivo, KIIIA presents an attractive starting point for the design of peptidomimetics. With the aim of improving potency and most importantly, subtype-selectivity profile, a number of KIIIA peptidomimetics have been recently synthesized. Strategies included stabilizing α helices with lactam bridges,104 introducing carboxamides to form hydrogen-bond-stabilized pseudo six-membered rings,105 adding N-substituting glycine residues, and synthesis of selenopeptide analogues.106 Although most of these peptidomimetics have a lower Nav1.7 activity than KIIIA and selectivity profiles that are still not adequate, they may serve as valuable templates for future leads. Another small µ-conotoxin, SIIIA107a has shown analgesic activity in rodent models of hyperalgesia, where it caused a reduction in acute and inflammatory nociceptive responses and blocked action potentials in sciatic nerves at 1 mM. SIIIA seems to be a Nav1.2-selective toxin but the exact mechanism of action through which it achieves analgesia remains unclear. Alewood and coworkers have recently reported a series of synthetic SIIIA analogues with a structurally minimized functional SIIIA scaffold that retained native functionality at Nav1.2.107b A µ-conotoxin, PIIIA,108a also selectively inhibits Nav1.4 and its mechanism has recently been examined in atomic detail.108b Using MD simulations Chen and 34 ACS Paragon Plus Environment

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coworkers have found that PIIIA binds to Nav1.4 in six different binding modes having similar energetics, a result consistent with a number of mutagenic studies. In all cases the toxin–channel complex was stabilized by three or four salt bridges. This finding suggested that other µconotoxins might also have multiple modes of binding to Navs. Inhibition studies with µconotoxins have suggested that Nav1.6 and Nav1.7 are the major contributors to peripheral nerve action potentials. Despite intense interest in Nav1.7 as a pain target, a selective conotoxin inhibitor of this subtype has not yet been found. Similarly, it would be useful to identify a conotoxin that selectively interacts with the Nav1.3 and/or Nav1.9 subtypes. Although very little is known about the functional activity profiles of all Nav subtypes for the different µ-conotoxins, Zhang and coworkers have recently quantitatively assessed the relative contributions of specific Nav subtypes to the TTX-sensitive INa of individual rat DRG neurons using three µ-conotoxins (TIIIA, PIIIA, and SmIIIA).108c Relatively little is known about the other four families of conotoxins that interact with Navs. µO-Conotoxins are Nav inhibitors that prevent activation by interacting with the voltage sensor of Navs in a site that remains to be fully defined and seems to partially overlap with Site 6 (Figure 4). In animal models of analgesia µO-conotoxins MrVIA and MrVIB109a showed an analgesic effect, which has been attributed to a relative selectivity for Nav1.8 over the TTXsensitive Nav subtypes expressed in DRG neurons.109b µO-conotoxins can also differentiate between Nav1.8 and Nav1.9 channels. Although both µ- and µO-conotoxins show promise as Nav antagonists to treat pain, µO-conotoxins are better positioned to be developed due their superior subtype-selectivity profile. As mentioned earlier, δ-conotoxins (25–35 residues) are agonists that extend action potentials when bound to Site 6 of Navs and cause a persistent neuronal firing by blocking the channel inactivation state. 35 ACS Paragon Plus Environment


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The first δ-conotoxin discovered was called the King Kong peptide because injection of the toxin into lobster caused the animal to continuously assume a dominant posture.110a Among the known δ-conotoxins, EVIA110b displayed selectivity for neuronal subtypes Nav1.2, Nav1.3, and Nav1.6 over Nav1.4 and Nav1.5. Another family of Nav conotoxin agonists, ι-conotoxin peptides are 40 to 50 residues in length and contain four disulfide bridges. ι-conotoxins shift Nav channel activation to more hyperpolarized potentials thereby causing these channels to open at voltages where they are normally closed. The receptor site to which ι-conotoxins bind remains to be determined. One interesting ι-conotoxin is RXIA,111a which at 50 µM potently activates Nav1.2, Nav1.6, and Nav1.7 subtypes without affecting other Nav subtypes.111b The µO§-conotoxin GVIIJ, the first discovered member of this recently identified class, contains an extra cysteine that forms a disulfide bond with an extracellular cysteine residue when blocking Nav channels.99

Scorpion toxins. Scorpions use a cocktail of toxins to immobilize their prey and to defend against predators. Scorpion venom consists of a complex mixture of peptides, enzymes, lipids, nucleotides, mucopolysaccharides, and biogenic amines. Within these cocktails, only a small set of peptides are responsible for the toxicity in humans and other mammals. For example, it has been shown that 90% of the toxicity of the Androctonus australis venom is due to only four peptidic toxins, AaHI to IV, accounting for only 2–3% of the weight of the crude venom obtained by electric stimulation. Scorpion toxins (ScTxs) block or modify the function of VGICs. Among these, long-chain peptides (61 to 70 amino acids) interfere with Nav channel voltage-sensing function, increasing the depolarization of the membrane and the release of neurotransmitters by affecting activation or inactivation states. There are two well-characterized groups of Nav-channel-specific toxins, α-ScTxs and βScTxs. While scorpions from North Africa contain α-ScTxs, those coming from North and South 36 ACS Paragon Plus Environment

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America possess β-ScTxs. α-ScTxs interact with Navs at Site 3 (Figure 4), slowing down the fast inactivation of the channel and prolonging the action potential, while β-ScTxs interact at Site 4 (Figure 4) and shift the voltage-dependent activation to more negative membrane potentials, leading to repetitive firing in muscles and nerves. Despite their different modes of action, α- and β-ScTxs display a number of common features. These neurotoxins consist of small basic proteins with a single chain of 61–70 amino acids, some of which are strictly conserved. They share the same general location for eight cysteine residues that form four similar disulfide bridges, so that they share a similar three-dimensional structure. While α-ScTxs induce painful responses, βScTxs are considered analgesic peptides.112 Recently, Durek and coworkers have synthesized and characterized OD1 (Figure 3F), an αScTx isolated from the venom of the Iranian yellow scorpion.113 OD1 is a potent modulator of mammalian Nav1.7 (EC50 = 4.5 nM), Nav1.4, and Nav1.6 and showed >1 µM selectivity over Nav1.2, Nav1.3, Nav1.5, and Nav1.8. Interestingly, Durek and coworkers also prepared nine OD1 mutants, of which one (triple mutant D9K, D10P, K11H) was 40-fold more selective for Nav1.7 over Nav1.6.113 Delmas and coworkers have recently isolated and pharmacologically studied αScTx Amm VIII and compared it with the classical α-ScTx AaHII.114 Amm VIII induced rapid mechanical and thermal pain hypersensitivities. Both toxins impair fast inactivation of Nav1.7 (AaHII EC50 = 6.8 nM and Amm VIII EC50 = 1.76 µM). Neither Nav1.8 nor Nav1.9 was affected by these toxins. Interestingly, because AmmVIII has a lower potency at Navs than AaHII, Amm VIII was devoid of toxicity when injected subcutaneously to mice. Other novel scorpion toxins such as rBmαTX47, a novel α-ScTx from the scorpion Buthus martensii Karsch, have recently been cloned and expressed by Wu, Y. and Li, T. who also highlighted the importance of expression vectors in affecting toxin pharmacology.115 37 ACS Paragon Plus Environment


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Unfortunately, only a few structure–function studies with scorpion toxins have been undertaken because of the challenges of obtaining multi-milligram quantities of these complex structures via isolation from natural sources or chemical synthesis. We envision that with the number of technological advances in the synthesis and isolation of structurally complex peptides, a new era of research in this fascinating field is just beginning.

Spider toxins. More than 42,700 species of spiders have been described and an even greater number remain to be characterized, making them the largest group of terrestrial predators.116 Spiders appear to have evolved a significantly larger repertoire of Nav channel toxins than any other venomous animal. Spider venoms are complex chemical cocktails that contain disulfide-rich peptides that modulate the activity of vertebrate Nav channels. Since a single venom can contain as many as 1,000 peptides, it has been conservatively estimated that more than 10 million bioactive peptides are likely to be present in the venoms of spiders, of which only 0.01% have been characterized. The only structures related to spider toxins that bind to Navs (NaSpTxs) are the δ- and µO-conotoxins. It is remarkable that two such taxonomically diverse animals, one confined to marine environments and the other a terrestrial predator, have evolved such similar molecular scaffolds for targeting the Nav channels of their prey. The mechanism by which NaSpTxs interact with mammalian Navs is multifaceted having three distinct effects on Nav channel function: inhibiting channel opening, delaying fast activation, and facilitating channel opening. There are 12 different families (named Family 1 to 12) of NaSpTxs based on the specific spider species where the toxin is found. NaSpTxs are useful therapeutic leads for the development of Nav-targeting analgesics. NaSpTxs contain an inhibitor cystine knot (ICK) motif that provides them with tremendous chemical, thermal, and biological stability.117 This is shown by the fact that NaSpTxs are stable in human serum for several days. (Notably, an 38 ACS Paragon Plus Environment

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ICK scaffold forms the core of the analgesic drug zinconotide (Prialt®), a peptidic toxin derived from the cone snail Conus magus. The drug targets calcium channels and is approved in the U.S. for the treatment of severe chronic pain. Because NaSpTxs are gating modifiers rather than pore blockers, NaSpTxs are more likely to display superior subtype selectivity compared with small compounds that bind in the highly conserved pore of the channel. Huwentoxins (HWTXs) are a group of neurotoxin peptides found in the venom of the Chinese bird tarantula Haplopelma huwena. Huwentoxin-2 (HWTX-II) is a 35-residue toxin that belongs to Family 1 SpTxs.118a HWTX-II inhibits the neuronal TTX-sensitive Navs at Site 4 in DRG cells and it showed selectivity at the hNav1.7 channel over rNav1.2, rNav1.3, rNav1.4 and hNav1.5. Recently, Yi and coworkers have reported that another huwentoxin, HWTX-IV shows antinociceptive effects in various mouse and rat models of inflammatory and neuropathic pain.118b Deng and coworkers have synthesized native HWTX-IV and three mutants (T28D, R29A, and Q34D). Substitution of three C-terminal residues resulted in a severe reduction of toxin binding affinities (10- to 200-fold) for TTX-sensitive Navs from DRG neurons, a result suggesting that these three residues may have critical interactions with TTX-sensitive Navs.119 The best-known exemplar of NaSpTxs is protoxin II (ProTx-II, Figure 3E), a 30-residue peptide with three disulfide bonds isolated from the tarantula Thrixopelma pruriens that docks at the DIIS3–S4 linker and traps the DIIS4 voltage sensor in the closed state (Site 4, Figure 4).120 ProTx-II is the most potent blocker of hNav1.7 reported to date (IC50 = 0.3 nM). ProTx-II is lethal to rats when administered intravenously (at doses ≥ 1 mg/kg) or intrathecally (at doses ≥ 0.1 mg/kg) because, although ProTx-II preferentially blocks Nav1.7 subtypes, it is also highly potent at Nav1.2, Nav1.5, and Nav1.6 subtypes (IC50 = 41 nM, 79 nM, and 26 nM, respectively). ProTx-II achieves Nav1.7 subtype selectivity probably due to the amino acid residue differences 39 ACS Paragon Plus Environment


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observed at the DIIS3–S4 extracellular loops of the different Nav subtypes (Figure 5B). Interestingly, some peptidic toxins have been observed to access their respective receptor sites and gain most of their binding affinity by partitioning into the lipid membrane. This seems to be the case for ProTx-II based on its demonstrated ability to bind to liposomes.121 Hainantoxin-IV (HNTX-IV) is a typical inhibitory cystine knot peptidic toxin that has been isolated from the venom of the Chinese bird spider Selenocosmia hainana.122 HNTX-IV selectively inhibits TTX-sensitive, but not TTX-resistant, Navs in adult DRG neurons. Recently, Wang and coworkers, while characterizing the activity of HNTX-IV on five different TTXsensitive mammalian Nav subtypes have observed that this toxin preferentially blocks the hNav1.7 subtype (IC50 = 21 nM), but also inhibits the rNav1.2 and rNav1.3 subtypes (IC50 = 36 nM and 375 nM, respectively) while showing little effect at rNav1.4 and hNav1.5 (at 1 µM 7.3% and 0% inhibition, respectively).123 Liang, S. and Liu, Z. have isolated the venom of the fishing spider Dolomedes sulfurous, and shown that it contains a number of neurotoxins that exhibit inhibitory effects on TTX-resistant Navs in rat DRG neurons.124 Liang and Liu’s recent work demonstrates the constantly evolving nature of spider venom research.

Other peptidic toxins. Centipedes form another class of ancient extant terrestrial arthropods that use venom to capture prey. Although centipede venoms have been largely neglected, Yang, King and coworkers have recently isolated µ-SLPTX-Ssm6a, a peptide from the venom of the Chinese red-headed centipede Scolopendra subspinipes mutilans that is a very potent and selective Nav1.7 inhibitor.59 µ-SLPTX-Ssm6a has a unique 46-residue amino acid sequence different from any known peptide or protein; it contains six cysteine residues that form three disulfide bonds. µ-SLPTX-Ssm6a potently inhibits human Nav1.7 (IC50 = 25 nM) and it is the most subtype-selective inhibitor reported to date (32-fold for Nav1.2 and over 150-fold for all 40 ACS Paragon Plus Environment

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other human subtypes). As discussed in the pain section of this review, µ-SLPTX-Ssm6a showed a potent analgesic effect in rodent models of nociception, being several times more efficacious than morphine in the mouse formalin-induced pain trial and equipotent to morphine in thermal and acid-induced-pain mouse models. Importantly, µ-SLPTX-Ssm6a showed no evident side effects on blood pressure, heart rate, or motor function at an intraperitoneal dose of 1 µmol/kg. Sea anemone venoms contain toxins such as anthopleurin B (Ap-B, Figure 3C)125a that are known to bind to Site 3 of Navs and can inhibit fast inactivation at nanomolar concentrations. Most of the biologically active peptides in sea anemones remain unexplored. Although a number of mutagenic and pharmacological studies have been performed with anemone toxins such as Ap-A, Ap-B, and ATX-II,125b research on this area is progressing slowly. A possible explanation for this is the complex molecular structure of the anemone toxins coupled with a usually observed higher activity at crustacean Navs and K+ ion channels. Zoanthids, marine animals found mainly in coral reefs and the deep sea, have been poorly studied and only some lowmolecular-weight compounds with anti-inflammatory and antiparasitic activity have been isolated. Recently, Lazcano-Pérez and coworkers assessing the biological activity of venom from the zoanthid P. caribaeorum on Nav1.7 channels, discovered a 3,648 kDa peptide that delayed Na+ current using the patch-clamp technique and rat cervical ganglion neurons.126 Although there have recently been major technological advances that facilitate highthroughput screening of venoms, as well as structural and functional characterization of venom components, many natural toxins remain to be discovered. In particular, the vast number of peptidic toxins represent an invaluable source for exploring how Nav subtype selectivity, a major challenge in this field, can be achieved. Despite the tedious nature of purifying neurotoxins from complex venom mixtures, researchers have been successful in identifying peptides with the 41 ACS Paragon Plus Environment


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potential to treat various human diseases. There are currently six FDA-approved drugs derived from venom peptides or proteins: zinconotide, a selective Cav2.2 channel inhibitor from the cone snail for the treatment of chronic pain; eptifibatide (Integrilin®) and tirofiban (Aggrastat®), two peptides from different snake species that target αIIIbβ3 integrin receptor for the treatment of acute coronary syndromes; bivalirudin (Angiomax®), a thrombin inhibitor from medicinal leech to treat coagulation during surgery; exenatide (Byetta®), a GLP-1 receptor agonist from the spit of the venomous lizard Gila monster to treat type 2 diabetes; and batroxobin (Baquting®), a blood factor Xa inhibitor to treat perioperative bleeding.127 The major technological advances mentioned previously, coupled with rapidly increasing understanding in other areas such as the nervous system, will advance the approval of more drugs that target VGICs, including Navs, in the near future.

CLINICAL UPDATE Most of the hundreds of ongoing clinical studies are evaluating new indications using firstgeneration Nav subtype-nonselective inhibitors that are already on the market, such as phenytoin, lidocaine, carbamazepine, bupivacaine, lamotrigine, oxcarbazepine, and eslicarbazepine, that bind at the local anesthetic site where amino acid residues are highly conserved among the different Nav subtypes (Figure 5A).1b This section focuses on the companies that have identified small-molecule Nav inhibitors with superior Nav subtype-selectivity profiles suitable for evaluation in clinical studies. Two patent application reviews regarding Nav inhibitors have recently been published.128


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Pfizer. After identification and characterization of 16 (Figure 9)56 as a selective Nav1.3 and Nav1.1 inhibitor (hIC50 Nav1.3 and Nav1.1 < 20 nM, Nav1.2 = 240 nM, Nav1.4, Nav1.5, Nav1.6, Nav1.7 and Nav1.8 > 10 µM), researchers at Pfizer discovered 17 (Figure 9), a selective Nav1.7 inhibitor.56 They have observed that both inhibitors (16 and 17) preferentially interact with the voltage-sensing region in DIV of Navs in the inactivated state and they have identified by mutagenesis studies three amino acid residues that contribute to the differences in their subtype selectivity and species potency of inhibitors 16 and 17.56 Optimization at the central phenyl ring of inhibitor 17 and its left portion, which is the part believed to play a crucial role in subtype selectivity, provided the compound PF-05089771 (structure not disclosed but possibly related to structure 18, Figure 9).129a PF-05089771 is a Nav1.7-selective inhibitor that has undergone twelve phase I studies to assess its efficacy, pharmacokinetics, safety, and tolerability in treating primary (inherited) erythromelalgia, postoperative dental pain, osteoarthritis (OA), and diabetic peripheral neuropathy (DPN). A phase II study with PF-05089771 for the treatment of DPN is ongoing.129b Pfizer continues to look actively for novel Nav1.7-selective inhibitors and has recently published three patent applications exemplified by 19,130 20,131 and 21132 (Figure 9). In addition to Nav1.7-selective inhibitors, Pfizer was developing PF-04531083 (structure not disclosed but possibly related to structure 22, Figure 9),133 a Nav1.8-selective inhibitor for the treatment of postsurgical dental pain; however, this study was not completed and PF-04531083 is no longer listed in the Pfizer pipeline. Pfizer. In addition, a benzimidazole series of potent Nav1.8 inhibitors (exemplified by structure 23, Figure 9) that possess a similar structure to Sumitomo Dainippon Nav1.7/Nav1.8 dual inhibitors (vide infra), has been recently described.134

Convergence Pharmaceuticals. Convergence, a company strongly focused on ion channels for the treatment of chronic pain, is developing CNV-1014802 (structure not 43 ACS Paragon Plus Environment


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disclosed)135 as a potent, and selective state-dependent Nav1.7 inhibitor. CNV-1014802 has received orphan-drug designation by the FDA and is being developed for the treatment of trigeminal neuralgia, a very severe form of facial pain. Results of a phase II study to evaluate the efficacy of CNV-1014802 for treating trigeminal neuralgia showed that this compound reduced the pain severity and the number of paroxysms in all primary and secondary outcomes (at a dose of 150 mg tid). Notably, this study demonstrated for the first time the efficacy of a selective state-dependent Nav1.7 inhibitor for treating chronic pain. This drug was well tolerated and showed no serious adverse events. Furthermore, recent results of a second phase II proof of concept study with CNV-1014802 (at a dose of 350 mg bid) in subjects with neuropathic pain from lumbosacral radiculopathy (sciatica), showed a statistically significant reduction in pain.135 According to the company web page, Convergence has two more Nav1.7-selective inhibitors, CNV-3000223 and CNV-3000164 (structures not disclosed),135b that are undergoing preclinical studies. In addition to drugs for chronic pain, Convergence is developing CNV-106436 (structure not disclosed),135b a highly potent and selective state-dependent Nav1.3 blocker for the treatment of epilepsy and bipolar disorders. There has been no disclosure of the chemical structure of any of these four Nav inhibitors, but based on a recent patent search they may be related to structures 24, 25, 26 and 27 (Figure 10).136,137

Xenon/Teva. Xenon and Teva are developing XEN-402 (also named TV-45070, structure not disclosed but possibly related to structure 28, Figure 11),138 a Nav1.7 inhibitor that has exhibited analgesia in a number of rat models of allodynia, such as neuropathic pain model of chronic constriction injury and a streptozotocin (STZ) model of diabetic neuropathy. A recent phase IIa study to evaluate the efficacy, safety, pharmacokinetics, and tolerability of XEN-402 showed that topical administration of XEN-402 (8% ointment strength bid for 14 or 21 days) was 44 ACS Paragon Plus Environment

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efficacious in reducing pain symptoms in primary/inherited erythromelalgia. Another phase II study to evaluate the efficacy, safety, pharmacokinetics, and tolerability of XEN-402 (8% ointment strength bid for 3 weeks) has been completed; however efficacy results have not yet been reported. Another clinical trial to evaluate XEN-402 in the topical treatment of primary OA of a single knee is currently recruiting patients and it is estimated that it will be completed by June 2015. XEN-402 received FDA orphan-drug designation as a pain drug in April 2013. Representative examples of recently published Nav1.7-selective inhibitors from Xenon (structures 29 and 30)139 and a Xenon/Genentech collaboration (structures 31 and 32) are exemplified in Figure 11.140 In addition to drugs for treating pain, Xenon is developing selective Nav1.6 inhibitors for the treatment of Dravet syndrome.141

Xenon/Genentech. A second product candidate, GDC-0276 (structure not disclosed),142 is being developed in collaboration with Genentech for the treatment of pain. In August 2014, Genentech received approval from Health Canada to initiate a Phase I clinical trial with GDC-0276.142

Sumitomo Dainippon Pharma. Sumitomo Dainippon Pharma is developing DSP2230 (structure not disclosed but possibly related to 33, Figure 12),143 a Nav1.7 and Nav1.8 dual inhibitor that exhibits antiallodynic effects in animal models of neuropathic pain; it is being evaluated in phase I studies in the U.K. and U.S.144 Sunovion has sponsored a phase I study to evaluate the analgesic efficacy of orally administered DSP-2230 in the human intradermal capsaicin and the ultraviolet-B models.145

Nektar Therapeutics. Nektar, a company specializing in water-soluble drug–polymer conjugates, is evaluating NKTR-171 in phase I trials.146 The structure of NKTR-171 has not been 45 ACS Paragon Plus Environment


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disclosed but could consist of a PEG–lidocaine or PEG–mexiletine conjugate such as N-mPEG8mexiletine, a conjugate that showed 66% reduction of writhing in an acetic acid writhing test in mice.147 NKTR-171 is being developed as an orally administered and peripherally restricted Nav inhibitor for the treatment of neuropathic pain. In preclinical studies, NKTR-171 demonstrated an improved efficacy and CNS side effect profile when compared to pregabalin. Additionally, at efficacious doses of analgesia, NKTR-171 exhibited a significantly reduced CNS penetration versus currently approved Nav inhibitors without showing major side effects such as impairment of motor coordination. In pre-clinical in vitro studies, NKTR-171 preferentially blocked abnormal rapidly firing neurons associated with neuropathic pain without affecting normal nerves, suggesting a frequency-dependent blockade of inactivated Navs.146

WEX Pharmaceuticals. WEX has two ongoing clinical studies to develop the marine natural product TTX,148 a phase III clinical study for the treatment of moderate to severe inadequately controlled cancer-related pain, and a phase II clinical study to treat chemotherapyinduced neuropathic pain in cancer patients. In both studies TTX is administered via subcutaneous injection twice a day for 4 days.148

AstraZeneca. AstraZeneca has recently evaluated 34 (AZD-3161, Figure 12) a Nav1.7 inhibitor in phase I trials for the treatment of peripheral neuropathic pain.149 Intradermal injections of 34 have been evaluated in normal subjects and in a model of ultraviolet-burned skin in healthy volunteers; however this compound is no longer listed in the AstraZeneca pipeline.

GlaxoSmithKline. GlaxoSmithKline is developing 35 (GSK-2339345, Figure 12),150 a broad-spectrum Nav inhibitor that is being evaluated in phase II clinical trials for the attenuation of cough by inhalation delivery in patients with chronic idiopathic cough.151 Compound 35 has 46 ACS Paragon Plus Environment

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exhibited efficacy by inhibiting citric-acid-evoked cough after intratracheal administration in guinea pig and dog models.152 In addition to drugs for treating cough, GlaxoSmithKline has been developing GSK-1014802 (structure not disclosed), a Nav antagonist in phase II studies for the treatment of bipolar depression.153

Gilead. Gilead is developing GS-6615 (structure not disclosed but possibly related to structure 36, Figure 12),154 a potent and selective inhibitor of the late sodium current of cardiac subtype Nav1.5 that has completed a phase I clinical study evaluating its effect on long QT-3 syndrome. An oral dose (10–60 mg over 12 h) of GS-6615 led to a shortening of the QTc interval in patients with long QT-3 syndrome without showing any safety concern. GS-6615 is also undergoing phase I clinical studies evaluating its potential for the treatment of hypertrophic cardiomyopathy and ventricular tachycardia/fibrillation.155

CONCLUSION Due to extensive recent research and discoveries in the field of Nav channels, today we have a substantial body of information available on channel structure, physiological roles of Nav subtypes and the impact of channel dysfunction on physiology leading to a variety of disorders. A huge number of publications underscores a high interest in this field which undoubtedly offers an enormous potential for discovering new therapies. Many attempts have been made to identify novel Nav channel modulators establishing a rich set of pharmacological tools to study channel structure and function and the effects of channel modulation in diseases. To date eight pharmacologically distinct ligand sites have been described on the pore forming Nav α subunits. The vigor of the current stream of research on Nav inhibitors is also highlighted by the hundreds 47 ACS Paragon Plus Environment


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of clinical studies currently underway to evaluate treatments for cardiovascular and respiratory diseases, epilepsy, and pain. The interest and need for novel pain therapies is particularly obvious. Pain affects 20% of the world’s population, and there is an estimated economic burden in the U.S. of $600 billion per year, which exceeds the combined annual cost of cancer, heart disease, and diabetes.156 Options available for the treatment of pain today suffer from limited efficacy and/or dose-limiting side effects. The current standard of care for moderate to severe chronic pain includes non-steroidal anti-inflammatory drugs (NSAIDs), tricyclic antidepressants, gabapentinoids and opioids. NSAIDs, as a widely prescribed option for OA, often suffer from minimal efficacy and potentially serious gastrointestinal and cardiovascular adverse effects. Gabapentinoids, such as gabapentin (Neurontin®) and pregabalin (Lyrica®) and tricyclic antidepressants show variable efficacy in pain patients and cause adverse effects, such as drowsiness, dizziness and weight gain. Opioids can cause serious side effects such as constipation, nausea, sedation, dizziness, vomiting, and respiratory depression, and may present a risk for addiction and tolerance in some patients.157 Notably, pain has emerged as a very promising therapeutic indication for Nav inhibitors selectively targeting Nav1.3, Nav1.7, Nav1.8, or Nav1.9 channels. In particular Nav1.7 stands out as a target to treat chronic pain, largely because humans who have no functional Nav1.7 channel suffer from chronic insensitivity to pain and those who carry Nav1.7 gain-of-function mutations suffer severe chronic pain syndromes. However, all currently approved Nav blockers in the clinic, such as local anesthetics, have limited clinical utility due to the lack of selectivity among the distinct Nav subtypes.


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The development of novel suitable therapeutic agents for selectively targeting Navs presents a number of challenges: (1) Navs are very complex molecular structures that rapidly transition through different states (closed, open, or inactivated). Ligand binding and functional activity often depends on the conformational state of the channel (state-dependence) which makes the assessment of structure-activity relationships challenging. (2) The high degree of amino acid sequence homology among the different Nav subtypes makes identifying or designing subtype-selective ligands extremely difficult. (3) Some Nav ligands show potency differences across animal species which can complicate the assessment of efficacy in preclinical in vivo models and the design of safety studies. A more detailed perspective on the molecular structure of Nav will help to overcome some of the above listed challenges. Several bacterial Nav crystal structures have been elucidated to date, providing a way to visualize these channels. We anticipate that eukaryotic Nav crystal structures containing bound ligands at different channel states will be solved in the near future. Combined with recent advances in molecular dynamics simulations this will enable the development of improved homology models for structure-based drug design. Additionally, the recent generation of stable cell lines overexpressing specific Nav subtypes, and the advancements in functional ionchannel screening technologies, such as high throughput automated patch clamp assay,158 have spurred the development of in vitro assays for the detection and characterization of selective state-dependent and state-independent channel modulators.159 Equipped with those tools researchers will be able to explore enormous collections of synthetic molecules and the remarkable diversity of venom-derived toxins eventually leading to the identification of subtypeselective Nav inhibitors for clinical validation. 49 ACS Paragon Plus Environment


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AUTHOR INFORMATION Corresponding author *Phone: 215-9938959. E-mail: [email protected] Notes The authors declare no competing financial interest.

Biographies Manuel de Lera Ruiz received his B.Sc. in Chemistry from the University Autónoma of Madrid, Spain, in 1997. After completion of his Ph.D. in 2001 from the University of Nottingham, U.K., he joined Professor Leo A. Paquette’s research laboratories as a postdoctoral fellow. In 2003, he started a career in medicinal chemistry at Schering-Plough Research Institute, and currently, he is an Associate Principal Scientist with Merck Research Laboratories in West Point, PA.

Richard L. Kraus received his Ph.D in Molecular Biology and Pharmacology from the University of Innsbruck, Austria in 1998. After doctoral and postdoctoral work in the field of ion channels in Professor Striessnig and Glossmanns’s laboratories, he joined the ion channel groups at Merck Research Laboratories in Rahway, NJ, and San Diego, CA. Richard Kraus is currently a Principal Scientist at Merck Research Laboratories in West Point, PA.


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ACKNOWLEDGMENTS The authors thank Dr. Deping Wang of Merck Structural Chemistry for his support with 3D models and graphic images, Sharon O’Brien and Sue Wilks of Merck Creative Studios for assistance with the figures, Dr. Christopher Burgey, Dr. Andrea Houghton, Dr. Mark Layton, and Dr. Cameron Cowden for their constructive comments and suggestions. Editing support from Philippa Solomon is greatly acknowledged. We also thank Dr. Jaume Balsells for all his encouraging support to put this manuscript together.

ABBREVIATIONS USED 3D, three-dimensional; Ap-A, anthopleurin-A; Ap-B, anthopleurin-B; ATP, adenosine triphosphate; bid, twice a day; BTX, batrachotoxin; C, cysteine; CAM, cell adhesion molecules; CFP, ciguatera fish poisoning; CNS, central nervous system; CTX, ciguatoxin; D, aspartic; DI– DIV, homologous domains I-IV; DEKA, aspartate-glutamate-lysine-alanine; DPN, diabetic peripheral neuropathy ; DRG, dorsal

root

ganglion;

EAE,

experimental

autoimmune

encephalomyelitis; EEDD, glutamate-glutamate-aspartate-aspartate; F, phenylalanine; GABA, gamma aminobutyric acid; GCG, (–)-gallocatechin-3-gallate; GEFS+, generalized epilepsy with febrile seizures plus; GLP-1, glugagon-like peptide receptor 1; GPCR, G protein-coupled receptors; H, histidine; HEK293, human embryonic kidney cells 293; HEK293T, SV40 T antigen expressing human embryonic kidney cells; hNav, human voltage-gated sodium channel; HNTX, hainantoxin; HWTX, huwentoxin; ICK, inhibitor cystine knot; IEM, erythromelalgia; Ig, immunoglobulin; INa, sodium current; K, lysine; kDa, kilodalton; kg, kilogram; L, leucine; LA, local anaesthetic; MDS, molecular dynamic simulations; mM, milimolar; MS, multiple sclerosis; 51 ACS Paragon Plus Environment


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MTSET, 2-(Trimethylammonium)-ethyl methanethiosulfonate-bromide; NMR nuclear magnetic resonance; NSAIDs, nonsteroidal anti-inflammatory drugs; NaSpTxs, spider toxins that bind to Navs; Nav, voltage-gated sodium channel; NavAb, voltage-gated sodium channel from the bacteria Arcobacter butzleri; NavMs, voltage-gated sodium channel from the bacteria Magnetococcus

sp;

NavRh,

voltage-gated

sodium

channel

from

the

bacteria

Alphaproteobacterium HIMB114; nM, nanomolar; OA, osteoarthritis; P, proline; PbTx, brevetoxin; PD, pore domain; PEPD, paroxysmal extreme pain disorder; PEG, poly(ethylene glycol); PGE2, prostaglandin E2; P-loops, pore extracellular loops; PNS, peripheral nervous system; ProTx-II, protoxin II; rNav, rat voltage-gated sodium channel; S1–S6, transmembrane helical segments; SAR, structure activity relationships; SCN, suprachiasmatic nucleus; ScTxs, scorpion toxins; SF, selectivity filter; SFN, small fiber neuralgia

; shRNA, small hairpin RNA;

SLPTX, scolopendra toxin; STX, saxitoxin; STZ, streptozotocin model of diabetic neuropathy; SVmab1, sodium channel voltage sensor monoclonal antibody 1; tid, three times a day; TTX, tetrodotoxin; µM, micromolar; VGIC, voltage-gated ion channels; VSD, voltage-sensing domain; VTD, veratridine; Y, tyrosine; ZTX, zetekitoxin.


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voltgage-sensistive sodium channel ligands brevetoxin and ciguatoxin. J. AOAC Int. 2014, 97 (2), 307-315.

FIGURE’S LEGENDS Figure 1. Nav channel architecture. (A) Topology of the human Nav channel α subunits. These proteins consist of four homologous domains DI (green), DII (pink), DIII (blue), and DIV (yellow) connected by intracellular linkers. Each domain contains six transmembrane helical segments (S1-6). Segments S1-4 form the voltage-sensing domain (VSD). Plus signs in S4 represent the positively charged voltage-sensing initiating protein (containing a number of arginine or lysine residues), whose movement leads to channel opening in response to membrane depolarization. Segments S5, S6 and the connecting pore-loops (P-loops) form the channel pore. The intracellular loop connecting DIIIS6 and DIVS1 functions as an inactivation gate closing the channel pore during fast inactivation. (B) Extracellular view of the open-channel conformation crystal structure of NavMs, a marine bacteria from Magnetococcus sp.12 Four identical domains are colored as in A to highlight parallels to the human Nav structure. (C) Side view of the openchannel conformation crystal structure of bacterial NavMs.12 Selectivity filter (SF) and activation gate are indicated at the center and intracellular part of the channel pore, respectively.

Figure 2. Tissue expression of Nav subtypes, and effects of Nav dysfunction on physiology.


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Figure 3. Toxins isolated from animal venoms that act on Nav channels. (A) Tetrodotoxin (TTX, 1) extracted from the puffer fish. (B) Batrachotoxin (BTX, 11) found in the skin of Panamanian golden frogs. (C), (D) and (E) Homology models of anthopleurin B, µ-conotoxin KIIIA, and ProTx-II extracted from sea anemones, cone snails, and tarantulas, respectively (based on the NMR structure of the heteropodatoxin HpTx2). (F) X-ray crystal structure of the α scorpion toxin (α-ScTx) OD1.

Figure 4. Location of eight Nav sites identified so far (six sites for different natural toxins plus the local anesthetic and ICA/PF binding sites). (A) Topology of the human Nav channel α subunit indicating binding sites. (B) and (C) An extracellular and side view, respectively, of the crystal structure of bacterial NavMs.12 Site 1 (red) is formed by the four P-loops and represents the binding site of pore blockers, such as TTX, STX, and µ-conotoxins. Site 2 (green) is the site for toxins such as BTX, VTD, and antillatoxin, which interact with S6 in DI and IV. α-ScTxs and Ap-B modulate Nav channels by binding to Site 3 (cyan) on the extracellular S3-S4 loop in DIV. Site 4 (blue) consists of the extracellular loops S1-S2 and S3-S4 of DII and constitutes the binding site of β-ScTxs, ProTx-II, and HWTX-II. Site 5 (pink), which comprises S6 of DI and S5 of DIV, has been identified as the interaction site for ciguatoxins and brevetoxins. Site 6 (orange), via S4 of DIV, binds µO- and δ-conotoxins. Site 7 (purple) is the local anesthetic binding site in the pore of the channels and consists of amino acid residues in S6 of DI, DIII, and DIV. Site 8 (yellow) is the ICA/PF site located in the VSD of DIV.



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Figure 5. Selected Nav α-subunit amino acid sequences involved in binding of selective and nonselective channel modulators. (A) Amino acid residues known to bind nonselective local anesthetics are conserved among Nav subtypes, and are highlighted in blue. (B) Amino acid sequences of extracellular loop S3-S4 in DII at Site 4 (highlighted in green). In contrast to local anesthetics, neurotoxin ProTx-II and antibody SVmab1 interact with DII-S3S4 and display Nav1.7 selectivity.

Figure 6. Structure of guanidinium-based non-peptidic toxins

Figure 7. Brevetoxins and ciguatoxins

Figure 8. Batrachotoxin, veratridine, (-)-gallocatechin-3-gallate & antillatoxin

Figure 9. Pfizer Nav inhibitors

Figure 10. Convergence Pharmaceuticals Nav inhibitors

Figure 11. Xenon/Teva/Genentech Nav inhibitors


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Figure 12. Other Nav inhibitors



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Figure 1. Nav channel architecture. (A) Topology of the human Nav channel α subunits. These proteins consist of four homologous domains DI (green), DII (pink), DIII (blue), and DIV (yellow) connected by intracellular linkers. Each domain contains six transmembrane helical segments (S1-6). Segments S1-4 form the voltage-sensing domain (VSD). Plus signs in S4 represent the positively charged voltage-sensing initiating protein (containing a number of arginine or lysine residues), whose movement leads to channel opening in response to membrane depolarization. Segments S5, S6 and the connecting pore-loops (P-loops) form the channel pore. The intracellular loop connecting DIIIS6 and DIVS1 functions as an inactivation gate closing the channel pore during fast inactivation. (B) Extracellular view of the open-channel conformation crystal structure of NavMs, a marine bacteria from Magnetococcus sp.12 Four identical domains are colored as in A to highlight parallels to the human Nav structure. (C) Side view of the open-channel conformation crystal structure of bacterial NavMs.12 Selectivity filter (SF) and activation gate are indicated at the center and intracellular part of the channel pore, respectively. 177x159mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Tissue expression of Nav subtypes, and effects of Nav dysfunction on physiology. 177x130mm (300 x 300 DPI)



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Figure 3. Toxins isolated from animal venoms that act on Nav channels. (A) Tetrodotoxin (TTX, 1) extracted from the puffer fish. (B) Batrachotoxin (BTX, 11) found in the skin of Panamanian golden frogs. (C), (D) and (E) Homology models of anthopleurin B, µ-conotoxin KIIIA, and ProTx-II extracted from sea anemones, cone snails, and tarantulas, respectively (based on the NMR structure of the heteropodatoxin HpTx2). (F) Xray crystal structure of the α scorpion toxin (α-ScTx) OD1. 177x174mm (300 x 300 DPI)


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Figure 4. Location of eight Nav sites identified so far (six sites for different natural toxins plus the local anesthetic and ICA/PF binding sites). (A) Topology of the human Nav channel α subunit indicating binding sites. (B) and (C) An extracellular and side view, respectively, of the crystal structure of bacterial NavMs.12 Site 1 (red) is formed by the four P-loops and represents the binding site of pore blockers, such as TTX, STX, and µ-conotoxins. Site 2 (green) is the site for toxins such as BTX, VTD, and antillatoxin, which interact with S6 in DI and IV. α-ScTxs and Ap-B modulate Nav channels by binding to Site 3 (cyan) on the extracellular S3-S4 loop in DIV. Site 4 (blue) consists of the extracellular loops S1-S2 and S3-S4 of DII and constitutes the binding site of β-ScTxs, ProTx-II, and HWTX-II. Site 5 (pink), which comprises S6 of DI and S5 of DIV, has been identified as the interaction site for ciguatoxins and brevetoxins. Site 6 (orange), via S4 of DIV, binds µO- and δ-conotoxins. Site 7 (purple) is the local anesthetic binding site in the pore of the channels and consists of amino acid residues in S6 of DI, DIII, and DIV. Site 8 (yellow) is the ICA/PF site located in the VSD of DIV. 177x167mm (300 x 300 DPI)



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Figure 5. Selected Nav α-subunit amino acid sequences involved in binding of selective and non-selective channel modulators. (A) Amino acid residues known to bind nonselective local anesthetics are conserved among Nav subtypes, and are highlighted in blue. (B) Amino acid sequences of extracellular loop S3-S4 in DII at Site 4 (highlighted in green). In contrast to local anesthetics, neurotoxin ProTx-II and antibody SVmab1 interact with DII-S3S4 and display Nav1.7 selectivity. 177x123mm (300 x 300 DPI)


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159x90mm (300 x 300 DPI)



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146x146mm (300 x 300 DPI)


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175x128mm (300 x 300 DPI)



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127x206mm (300 x 300 DPI)


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130x90mm (300 x 300 DPI)



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129x171mm (300 x 300 DPI)


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152x97mm (300 x 300 DPI)



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208x53mm (300 x 300 DPI)


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Voltage-Gated Sodium Channels: Structure, Function, Pharmacology

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