Scorpion Toxins from Buthus martensii Karsch (BmK) as Potential Therapeutic Agents for Neurological Disorders: State of the Art and Beyond

Scorpions are fascinating creatures which became residents of the planet well before human beings dwelled on Earth. Scorpions are always considered as a figure of fear, causing notable pain or mortality throughout the world. Their venoms are cocktails of bioactive molecules, called toxins, which are responsible for their toxicity. Fortunately, medical researchers have turned the life-threatening toxins into life-saving therapeutics. From Song Dynasty in ancient China, scorpions and their venoms have been applied in traditional medicine for treating neurological disorders, such as pain, stroke, and epilepsy. Neurotoxins purified from Chinese scorpion Buthus Martensii Karsch (BmK) are considered as the main active ingredients, which act on membrane ion channels. Long-chain toxins of BmK, composed of 58–76 amino acids, could specifically recognize voltage-gated sodium channels (VGSCs). Short-chain BmK toxins, containing 28–40 amino acids, are found to modulate the potassium or chloride channels. These components draw attention as useful scaffolds for drug-design in order to tackle the emerging global medical threats. In this chapter, we aim to summarize the most promising candidates that have been isolated from BmK venoms for drug development.


Introduction
Recent advances underlying medical studies have illuminated that several neurological disorders such as epilepsy, chronic pain, multiple sclerosis, stroke, brain tumor etc. are induced by dysfunction of membrane ion channels [1][2][3]. Up to now, multiple drugs specifically targeting ion channels have been designed to treat the diseases [4]. Some clinical studies and trials have also been initiated to discriminate therapeutic potentials of natural toxins and their derivatives such as scorpion toxins, spider toxins, snake toxins, sea anemone toxins, and toad venom, which could recognize relevant ion channels [5]. peptides, scorpion toxins are classified into long-chain toxins and short-chain toxins. The long-chain scorpion toxins composed of 58-76 amino acid residues mainly act on voltage-gated sodium channels (VGSCs), while the short-chain scorpion toxins containing 28-40 amino acid residues generally target K + or Cl − channels [9] ( Figure 1B). Based on their physiological effects on VGSC gating and binding properties, the long-chain toxins can be further classified into two categories: α-toxins, such as BmK I, a 64-residue α-like toxin isolated from BmK [2], and BmK αIV, a novel cloned 68-residue polypeptide, binding to neurotoxin receptor site 3 of the VGSC, with inhibitory effects on the fast inactivation of VGSCs (Figure 2). β-toxins, which bind to receptor site 4 such as BmK IT2 as well as BmK AEP, two 64-residue inhibitory β-toxins [10], and BmK AS, a 66-residue β-like toxin, could shift the threshold of VGSCs activation to more negative membrane potentials [11][12][13][14] (Figure 2). By sequence alignment and phylogenetic trees, it could be found that the primary structure of BmK I is similar to that of the classical α-like toxin Lqh III, while the structural properties of BmK αIV are more similar to that of the classical α-toxin AaH II ( Figure 1C). In addition, the structure of BmK IT2 and BmK AEP are similar to that of the classical β-toxin Lqh IT2, but it is quite different from the structure of BmK AS which is also separated from BmK ( Figure 1C). Among short-chain toxins, martentoxin and BmP02 are considered as the specific blockers of BK channel (α + β4) and Kv1.3, respectively [15][16][17]. From sequence alignment and phylogenetic trees, martentoxin have low homology with the classical BK channel blocker charybdotoxin (ChTX), isolated and purified from Structure of VGSC and its pharmacological characterization modulated by α/α-like or β/β-like scorpion toxins. Schematic representation of Nav channels' α and β subunits. The α subunit of Nav channels is illustrated along with not only β1 but also β2 subunits; the β subunits' extracellular domains are exhibited to be an immunoglobulin-like fold that interacts with the loops in α subunits. Leiurus quinquestriatus, but have high homology with Lqh 15-1 (ChTX2), another BK channel blocker from Leiurus quinquestriatus (Figure 1B and C). BmK CT could recognize the glioma-specific chloride channels. In this chapter, we aim to describe the most promising candidates for drug development that have been isolated from BmK venoms, with categorization according to their biological activity.

Analgesic effects of BmK toxins against VGSCs
Pain seriously damages human health and quality of life, so it is of importance to find effective analgesic targets and drugs. Nav channels (VGSCs) are transmembrane proteins responsible for generation and conduction of APs (action potentials) in excitable cells [1][2][3]. Of the nine functional α subunits (Nav1.1-1.9), Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are distributed in primary sensory neurons, playing a crucial role in nociception and chronic pain [4]. In detail, mechanical pain is mainly caused by Nav1.6 [5]. Functional acquired mutations in Nav1.7 cause severe thermal hyperalgesia [6], while Nav1.7 with loss-of-function mutations leads to pain insensitivity [7]. Nav1.8 contributes to the APs generation in peripheral system and Nav1.9 plays a role in persistent Na+ currents in small-diameter dorsal root ganglia (DRG) neurons [8] BmK AS, polypeptide composed of 66 amino acid residues purified from BmK venom, was a unique β-like scorpion toxin with many distinct functions [41]. In the peripheral nervous system (PNS), BmK AS-induced antinociceptive effect on inflammation-induced thermal as well as spontaneous pain and mechanical hyperalgesia [12,42]. Peripheral or spinal delivery of BmK AS significantly suppressed formalin-induced nociceptive behaviors and c-Fos expression in spinal cord [12,43]. In order to clarify the mechanisms underlying antinociceptive effects of receptor site 4 toxins on VGSCs, the primary sensory neurons (dorsal root ganglion, DRG) isolated from the L4-L6 of adult rats are usually chosen for investigation. Patch clamp recording showed that BmK AS could significantly decrease excitability of small DRG neurons, by depressing the peak tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) Na + currents of DRG neurons, and causing a negative shift of voltage-dependent activation [12]. Furthermore, BmK AS reduces the peak currents, facilitates steady-state activation, and inhibits slow inactivation of the Nav1.3 channels [44]. Through testing the VGSCs endogenously expressed in the DRG neuroblastoma ND7-23 cells as well as heterologously expressed Nav1.2 in Xenopus oocytes, it exhibited a U-shaped modulation of gating kinetics by BmK AS over a wide range of concentrations. BmK AS could suppress the peak currents, facilitate steady-state activation of VGSCs endogenously expressed in ND7-23 cells, while it did not affect the voltage-dependent activation and persistent currents of Nav1.2 [45]. These results provide a better understanding of the peripheral antiinjury sensation of BmK, which selectively inhibited the activity of Nav1.3 and DRG subtypes of VGSCs.
BmK IT2, consisting of 61 amino acid residues, contains 4 disulfide bonds, and could induce strong insect toxicity [10]. Like other depressant toxins, such as LqhIT2 [46,47], BmK IT2 possesses two non-interacting binding sites (the high/ low-affinity binding sites) on insect nerve membranes [48,49]. But a previous study also found that formalin-induced spontaneous pain behavior and spinal c-Fos expression could be effectively suppressed by either pre-or post-treatment with intrathecal BmK IT2 [50], which strongly implied that BmK IT2 could not only bind to insect VGSCs, but also recognize mammal VGSC subtypes. In fact, the inhibition of BmK IT2 on total Na + currents was observed in small DRG neurons [13]. By testing VGSC subtypes in Xenopus oocytes expression system, Nav1.2, Nav1.3, and Nav1.6 display insensitive property to BmK IT2, suggesting that other isoforms, especially Nav1.7-1.9, might be involved in the suppressive activity of BmK IT2 in rat pathological models [51]. The results illuminated that BmK IT2 can be developed as a novel analgesic peptide with therapeutic potential.

Antiepileptic activity of BmK Na + channel toxins
VGSCs play a critical role in the generation and propagation of neuroexcitibilty. Genetic alterations in VGSC genes are considered to be associated with epileptogenesis. The SCN1A (Nav1.1 gene) is the most relevant VGSC gene for epilepsy in clinical tests. More than 1200 Nav1.1 mutants have been characterized to be associated with epilepsy, most of variants mutations lead to febrile seizures [52]. Nav1.2 subunits are mainly distributed in the Ranvier node and axon-initiating segment (AIS). The mutation of Nav1.2 (SCN2A) is relevant to various epilepsies, such as Dravet's syndrome (DS), benign familial neonatal seizures (BFNIS), hereditary epilepsy with febrile seizures plus (GEFS+), and other stubborn childhood epileptic encephalopathies [53]. Another VGSC subtype widely distributed in CNS is Nav1.6, which is mainly distributed to the soma and synaptic origins. Mutations of Nav1.6 could induce severe epileptic encephalopathy exhibiting autistic features, early onset seizures, intellectual disability, ataxia, or sudden unexpected death in epilepsy (SUDEP) [54]. Therefore, this evidence strongly implies that natural products inhibiting VGSCs could also have the potential for suppressing the epileptic seizure.
BmK AEP, composed of 61 residues with 4 disulfide bonds, is the first antiepilepsy peptide purified from scorpion venom. BmK AEP was less toxic to mice and insects, while it had forceful anticonvulsant effects on epileptic rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP) [55]. BmK AEP has been reported to display anti-epileptic activity in a coriaria lactone-induced epileptic model in the rat with comparable efficacy to diazepam [56]. Recent studies demonstrated that BmK AEP concentration-dependently suppresses the Na + currents of Nav1.3 and Nav1.6, heterologously expressed in HEK293 cells, and shifts the voltage-dependent activation to the hyperpolarized direction, with minimal effects on steady-state inactivation [14].
Through intrahippocampal injection, β scorpion toxin BmK AS produced obviously anticonvulsant activity on the pentylenetetrazol (PTZ)-induced epileptic rodents. It could not only suppress the duration and number of high-amplitude, high-frequency discharges (HAFDs) in electroencephalography (EEG), but also obviously reduce the peak Na + currents of hippocampal pyramidal cells [57,58]. By contrast, BmK AS did not regulate the epileptiform EEG of pilocarpine model over the same dose range [57]. Intrahippocampal injection of BmK AS obviously reduced the increase of c-Fos expression evoked by pilocarpine, implying that neuronal hyperactivity is decreased during the epileptic state [43].
Injection of BmK IT2 at hippocampal CA1 region could dose-dependently inhibit PTZ-induced epilepsy-like behavior as well as reduce the number and duration of HAFD on PTZ-induced epileptic EEG components. Similarly, BmK IT2 significantly prolonged the incubation period of status epilepticus (SE) onset, reduced the severity of SE, and inhibited the expression of c-fos in the hippocampus during SE of pilocarpine-induced epileptic rodents [59]. BmK IT2, which relieves epileptic symptoms, is thought to inhibit the activity of VGSC subtypes. Binding experiments showed that BmK IT2 could recognize neuronal synaptosome membranes. The patch-clamp experiment also proved that BmK IT2 can inhibit the persistent sodium current of hippocampal pyramidal neurons [59]. However, previous studies have found that BmK IT2 had no significant inhibitory effect on the peak Na + currents of Nav1.2, 1.3, and 1.6 heterologous-expressed in oocytes [51]. It is suggested that BmK IT2 might act on Nav1.1 or Nav1.7 in the central nervous system (CNS).

Antiepileptic activity of BmK K + channel toxins
BK channels, widely expressed in CNS, are voltage-and Ca 2+ -activated K + channels with large conductance [60][61][62] (Figure 3). They have been shown to modulate fast afterhyperpolarization (fAHPs) and rapid spike repolarization in a number of types of neurons [63][64][65]. Under pathological state, it interacted the inactivation of Nav channels, with inducing neuronal spike shortening and increasing in firing rate as well as excitatory transmitter release, which could exacerbate seizure bursts [66][67][68].
Pentylenetetrazol (PTZ)-induced generalized tonic-clonic seizures give rise to a BK channel gain-of-function, characterized by increased BK currents as well as neuronal firing in the somatosensory cortex [70]. Interestingly, the BK channel blocker, paxilline, suppressed generalized tonic-clonic seizures in picrotoxin or  PTZ-induced epileptic animal models, and reversed the elevated neuronal firing which follows tonic-clonic seizures [70,71].
Martentoxin, a polypeptide consisting of 37 residues isolated from the venom of BmK, could selectively block iberiotoxin-insensitive BK channel subtye (α + β4) [15,72], with no obvious effects on BK channels with α subunit alone. In animal model experiments, martentoxin could prolong the latency and decrease the duration, as well as seizure numbers, especially the high stage seizure, of seizures induced by PTZ. The attitude and the duration of epileptic discharge are both decreased by intra-hippocampal injection with martentoxin [73] (Figure 4).

Anti-multiple sclerosis effects of BmK Kv1.3 blockers
Multiple sclerosis (MS), a neuroinflammatory demyelinating disease, is the second most common neurological disease. The occurrence of multiple sclerosis is often accompanied by the destruction of the blood-brain barrier (BBB) and the infiltration of the central nervous system by reactive T cell [74]. These cells rapidly produce large amounts of pro-inflammatory cytokines, such as IFN-γ and IL-4, inducing sebaceous lesions or damage by targeting myelin basic proteins, thereby promoting shedding.
The potassium channel Kv1.3 was first discovered in human T-cells in 1984 [75]. Accumulated data display Kv1.3 in myelin-reactive T cells from the peripheral blood (PB) underlying MS patients is more highly expressed compared with healthy people [76]. In animal model of experimental autoimmune encephalitis (EAE), it has also been confirmed that the expression of Kv1.3 is significantly elevated [77]. Kv1.3 blocks membrane depolarization and maintains the driving force for Ca 2+ entry by effluxing K + , which in turn participates in T cell activation, Ca 2+ activation signaling cascade, leading to T cell proliferation and cytokine production [78,79]. These findings make Kv1.3 a valuable potential therapeutic target for immunosuppression in MS and EAE [80]. The therapeutic efficacy of Kv1.3 channel blockers has been evidenced by not only in in vitro assays on suppressing cytokine secretion and the proliferation of T cells, but also by in vivo experiments on diverse animal models of autoimmune diseases [81][82][83][84][85].
A variety of animal toxin peptides have been found to have the same channel target. The venoms of different species such as scorpions, anemones, snakes, and cone snails constitute a peptide damper for Kv1.3 [86][87][88]. Studies have shown that different toxin peptides have different affinities for the Kv1.3 channel and can inhibit Kv1.3 in the picomolar to nanomolar range [89].
BmKTX is an α-KTx toxin purified from the venom of BmK with 37 amino acids, which has an amidated C-terminal, and blocks Kv1.3 current with nanomolar concentration [90,91]. However, in addition to being selective for Kv1.3, BmKTX also has affinity for other K+ channels, which promotes the design as well as appearance of highly selective BmKTX structural analogs [92]. The BmKTX D33H variant was produced by replacing the Asp33 residue with His in BmKTX. The selectivity of this novel BmKTX analog is 10,000-fold higher than wild-type BmKTX for targeting Kv1.3 [92,93]. ADWX-1, a novel peptide based on the scorpion toxin BmKTX, replaces three residues of BmKTX (Gly 11, Ile 28, and Asp 33) with Arg 11, Thr 28, and His 33. The ADWX-1 peptide not only has a picomolar affinity (IC 50, 1.89 pm) for blocking Kv1.3, but its activity is increased 100-fold compared to the native BmKTX toxin [94]. More importantly, ADWX-1 also showed good selectivity on Kv1.3 compared to the related Kv1.1 and Kv1.2 channels. The data show that both BmKTX-D33H and ADWX-1 can effectively inhibit the activation and subsequent proliferation of human and rat CD4 + CCR7-TEM cells and the secretion of cytokines [93,94]. It is similar to the pharmacological properties of ShK-186, an anemone toxin analog that has been used in clinical research as a novel drug for the treatment of autoimmune diseases [95]. In addition, ADWX-1 can selectively inhibit the activation of effector memory T cells by inhibiting Kv1.3, thereby significantly improving the symptoms of experimental autoimmune encephalomyelitis (EAE) in a rat model [84,93]. The results above illuminated that BmKTX-D33H as well as ADWX-1 have the potential for clinical treatments of Kv1.3-related channel diseases.
BmP02, also referred to α-KTx9.1, is a short peptide toxin from the BmK scorpion. It is comprised of 28 amino acids, whose tertiary structure is stabilized by 3 disulfide bonds [36,96]. It was found that it has nanomolar affinity for Kv1.3 [17,97]. Functional characterization of BmP02 as a highly selective and potent Kv1.3targeted peptide will help develop novel therapeutic agents for human autoimmune diseases.

Anti-stroke potential of BmK Kv1.3 blockers
Stroke is an acute cerebrovascular disorder that causes brain tissue damage, which is the second leading disease causing sudden death after ischemic heart disease and accounts for 9% of deaths worldwide [83]. Ischemic stroke is the most common type of stroke, usually occurring when the blood vessels in the neck or brain are blocked [98]. In the early stages of stroke, activated macrophages or microglial cells (M1 type) release a variety of inflammatory factors (TNF-α, IL-1-β, IL-23), trigger neuronal damage, and induce TEM cell-mediated further inflammatory responses [99]. A few days later, macrophages could change to M2-like functions, begin to clear various inflammatory factors, cell debris, and secrete anti-inflammatory as well as neurotrophic factors (IL-10, TGF-β, IGF-1) to promote injury recovery [99].
Kv1.3 plays important roles in microglia as well as macrophage activation by modulating Ca 2+ signaling, oxidative burst, cytokine production, and neuronal killing [100][101][102], which is required for microglia or macrophage M1-like proinflammatory activation in vivo [103]. Activated microglia in the pathology of ischemic stroke significantly contributes to secondary expansion of the infarct, and Kv1.3 blockers are thought to be useful in ameliorating this condition [104,105]. Studies have shown that while Kv1.3 inhibitors preferentially inhibit "M1-like" inflammatory microglia/macrophage functions they can preserve beneficial "M2-like" functions [106,107].
BmP02 and BmKTX act as BmK K + channel toxins that can effectively inhibit Kv1.3. We speculate that they and their derivatives may also reduce pro-inflammatory factors and improve brain damage by inhibiting the M1-like function of microglia or macrophages.

Anti-glioma activity
Glioma shows the general characteristics of tumor cells, with the difference being that the specific chloride channel current (CCC) is a unique electrophysiological feature of glioma cells. The current intensity always increases with the increase of malignant degree of glioma [108]. The specific type of chloride channel on glioma cells can regulate the morphology and volume of cells, which are involved in the process of tumor cell proliferation and metastasis. Abnormal expression of chloride channel currents in glioma could be regarded as a kind of chloride channel disease, especially in glioma with high malignancy [109]. Therefore, it may provide a novel idea for the diagnosis and treatment of glioma by blocking its specific chloride channel current, from the perspective of ion channel disease.
BmK chloride channel toxins, BmK CT, are short-chain neurotoxin proteins composed of 36 amino acids and contain 4 pairs of disulfide bonds, which have 68% homology with chlorotoxin (CTX), a chloride channel toxin isolated from scorpion Leiurus quinquestriatus. BmK CT could not only specifically block the glioma chloride channels, but also recognize the matrix metalloproteinases-2 (MMP-2) for inhibiting glioma migration [110,111]. The recombinant protein GST-BmK CT significantly suppresses on tumor growth in nude mice, with an inhibition rate of 86% in vivo. The tumor metastasis in the lung lesion area was only 38% in the BmK CT-treated group compared to 75% in the control group [112]. In addition, BmK CT could promote the sensitivity of chemotherapeutic drug temozolomide-induced cell apoptosis of glioma U251 cells in vitro, which is through inhibiting the AKT signaling pathway [113]. On the one hand, the specific inhibition of the proliferation and metastasis of glioma cells suggests BmK CT as an ideal candidate to treat glioma. On the other hand, due to the abundant expression of chloride channels in glioma cells, BmK CT is also used for imaging and treating glioma by conjugating it with Cy5.5, FND, or 131I/125I [114].

Proposal
Up to now, there are 15 venom-derived drugs used to treat a variety of diseases, including hypertension, pain, and diabetes, in clinic. As a result, many lives have been saved. In addition, 13 animal-derived toxins are considered to be drug candidates, and have entered clinical trials [115]. Among them, scorpion toxin chlorotoxin, isolated from Leiurus quinquestriatus, is under phase II clinical trial. It was reported that Iodine-131-chlorotoxin (TM-601) is a targeted drug candidate for the treatment of gliomas because it could cross the blood-brain as well as some tissue barriers and specifically bind to malignant brain tumor cells without influencing the function of normal cells [116]. ShK derivatives, ShK-186 and ShK-192, are mainly used to treat autoimmune diseases, including neuroinflammatory multiple sclerosis by targeting Kv1.3 channels. In this review, we discuss the possibility of BmK scorpion toxins for clinical treatment on ion channel-relevant neurological disorders. It is shown that long-chain scorpion toxins, such as BmK IT2 and BmK AS, could effectively suppress neuroexcitability in nociception and epileptic seizure via VGSCs. In vivo study demonstrated that inhibition of Kv1.3 is favorable for the reversion of neuroinflammatory diseases by BmKTX and BmP02. It is also found that BmK CT could specifically suppress proliferation as well as metastasis of glioma cells. This brings the dawn to the effective control of neurological diseases suspected of overcoming, such as chronic pain, MS, intractable epilepsy, and giloma.
However, it is still a challenge for BmK toxins used to the treatment of neurology disorders. The first problem underlying the application of these peptides is that they could not be taken orally, mainly because they are difficult to penetrate the intestinal mucosa. Due to their molecular size, polarity, hydrophilicity, and chargeability, the cell membrane penetration of BmK toxins is hampered. The second obstacle is that BmK toxins cannot cross the blood-brain barrier. Different from multiple sclerosis, the myelin and blood-brain barrier are not destroyed in other neurological diseases [117]. Clinical application of BmK toxins for treating these diseases will encounter difficulties. Fortunately, the situation is not unsolvable, we still have a glimmer of light. A few years ago, scientists at the Sunnybrook Health Science Center in Canada used focused ultrasound technology to successfully pass chemotherapy drugs across the blood-brain barrier in a non-invasive manner [118] and reach the location of the tumor, which is of great significance in the field of neuropharmacology. In addition, the cell penetrating peptide (CPP) [119] with a strong cell membrane penetration, could be used as a drug carrier to assist the passage of polypeptide drugs across the cell membrane [117]. The fusion protein consists of CPP and BmK toxin might be developed as an oral drug for treating neurological disorders. In short, finding suitable, safe, and efficient ways to promote the clinical use of BmK toxins are most valuable points to be solved.