**4. Antiepileptic and neuroprotective effects**

small peptide consisting of nine amino acid residues, Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which exhibits a high degree of homology with bradykinin (BK), except for the substitution of Thr for Ser in position 6 at BK. As a result, small changes in their secondary structures are observed [74]. This modification has been regarded as responsible for increas‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical


activating presynaptic B2 receptors, which activate descending adrenergic pathways. Studies investigating the role of kinins in the CNS provide new information on the supraspinal sys‐ tem of the pain control, whose modulation may represent a new strategy to control pain-

**Figure 1.** Tridimensional structure of antinociceptive peptides isolated from arthropod venoms. (A) BMK IT2; (B) HWTX 1; (C) ω-Agatoxin IVA; (D) π-Theraphotoxin-Pc1a; (E) Mellitin. Uniprot entry code: P68727, P56676, P30288,

Besides peptides, some studies have evaluated the analgesic activity of acylpolyamines that can be used as new alternative drugs for the treatment of chronic pain, as well as tools for the study of the functional role of the AMPA/kainate receptors in the processing of nocicep‐ tive pain [77]. In this regard, intrathecal administration of different doses of these toxins blocked thermally induced allodynia [78] and hyperalgesia [79]. The effect of these neuro‐



ing B2 receptor affinity and potency of Thr6

phine and 4 times more potent than BK in tail-flick test. Thr6

Thr6

Applications

98

related pathologies [76].

P60514 and P01501, respectively.

Neurodegenerative disorders comprise a wide range of conditions mostly characterized by a progressive loss of neuronal function and neuronal cell death. The incidence of these diseas‐ es in population differs greatly. In conditions such as Parkinson disease and Alzheimer, the number of cases significantly increases in elderly, whereas epileptic patients are mostly chil‐ dren and adolescents. Many processes may trigger neuronal cell death, such as trauma, stroke, tumors, infections, genetic factors and biochemical alterations. Among the latest, the alterations in Ca2+-mediated signaling is thought to play a key role in many neurodegenera‐ tive disorders and the increase in intracellular Ca2+ concentration might alter neuronal mem‐ brane potential [82]. Moreover, the hyperactivation of excitatory transmission mediated mostly by L-glutamate and its ionotropic receptors; kainate, AMPA and NMDA, is responsi‐ ble for the excessive cationic influx that depolarizes neuronal cells and lead to sustained hy‐ perexcitation observed in brain pathologies such as epilepsy [83]. This increase in glutamatergic activity often referred to as glutamate excitotoxicity [84], might also involve non-receptor neurochemical events such as failure in glutamate uptake system, which ends with an increase in the availability of this neurotransmitter in the synaptic cleft [85,86]. The importance of L-glutamate in neurological disorders relies on the fact that this neurotrans‐ mitter is release in the great majority of fast synapses in CNS [84,83]. In this context, many molecules mostly peptides and acylpolyamines, acting on ion channels, receptors and trans‐ porters were isolated from arthropod venoms, remarkably spiders, scorpions and wasps [3]. According to [82], polyamines are non-specific antagonist of ligand-gated ion channels, act‐ ing at glutamatergic and Ach receptors in an uncompetitive way, that is, the receptor must be activated in order to occur the blockade. This mode of action might diminish the side ef‐ fects of newly designed medicines, since it blocks only the activated receptors, but does not prevent their opening.

The venom of the orb-web spider *Nephilia clavata* was one of the first venoms studied during the 80s, which resulted in the identification of small compounds named acylpolyamines, among whose we may find jorotoxin (JSTX), one of the first glutamate receptor uncompeti‐ tive antagonists [83,84]. Together with JSTX, another polyamines such as argiopin from the venom of the spider *Argiope lobata* [85] and philantotoxin (PhTx) from the venom of the soli‐ tary wasp *Philanthus triangulum* [86]. Following the structural characterization and studies in insect or crustaceans, the reports on the action of these polyamines in mammalian CNS started to take place, mostly during the 90s [87]. JSTX-1 and JSTX-3 are synthetic analogues of JSTX. The first inhibits kainate-induced seizures, whereas the latter block glutamate re‐ lease and hippocampal epileptic discharges [88,89]. Later, JSTX-3 was shown to inhibit the formation of superoxide dismutase-1 (SOD-1) aggregates that lead mutant motor neurons (mSOD-1) to death during the familiar form of the neurodegenerative disease, amyotrophic lateral sclerosis [90]. The authors concluded that increased Ca2+ influx mainly through AM‐ PA/kainate glutamate receptors make mutant neurons more vulnerable to damage and therefore, JSTX-3 is an interesting neuroprotective agent in this model.

The African tarantula *Hysterocrates gigas* known as the giant baboon spider, inhabits the rain forests of West Africa. The isolation of the venom of *H. gigas*, resulted in the identification of

New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

The arboreal tarantula *Psalmopoeus cambridgei* is an aggressive spider that lives in the tropi‐ cal forests of Trindad. As mentioned before, PcTx-1 (π-theraphotoxin-Pc1a) present in *P. cambridgei* venom is the only gating modifier of ASICs [50]. In addition to pain inhibitor, it exerts an interesting neuroprotective and a possible antidepressant activity due to the in‐ volvement of ASICs in cell excitability. A drop in pH from neutral 7.4 to more acid extracel‐

In the light of these facts, Yang and coworkers [105] investigated the neuroprotective activi‐ ty of PcTx in neurons from newborn piglets submitted to a model of asphyxia-induced car‐ diac arrest. Data show that the administration of PcTx before the hypoxia-ischemia insult partially prevents the death of neurons in putamen, the most vulnerable encephalic area in this model. The addition of MK-801, a NMDA antagonist, in combination with PcTx exerted better results in cell survival, but in low doses of MK-801. In addition to protection of neuro‐ nal cells, treatment with PcTx accelerated neurologic recovery. These results point PcTx as a very unique neurotoxin that should be used as tool in the investigation of processes under‐

Bees and wasps are part of the group of the insects, whose stings release a cocktail of toxins, including enzymes, peptides and biogenic amines [106]. Toxins in bee venom have received attention for their properties as anti-inflammatory agents, and in many countries, physicians even prescribe bee stings as treatment of rheumatologic diseases. Recently, Doo and collea‐ gues [107] showed that the bee venom when injected in rats with induced Parkinson disease

Regarding solitary wasps, the most studied wasp species is the European beewolf, *Philan‐ thus triangulum*, the natural predators of honeybees. The adult individuals of this species are herbivores, whereas the larvae eat the paralyzed bees brought to the colony by foraging wasps. The isolation of venom contents begun in the early 80s and revealed that among oth‐ er classes of molecules, *P. triangulum* venom contains potent acylpolyamines [86]. Philantho‐ toxins, like other acylpolyamines are mostly potent and selective antagonists of vertebrate and invertebrate glutamate receptors, particularly AMPA receptors [108]. The first isolated and most studied philathotoxin is PhTX-433 and its synthetic analogue, PhTx-343, which an‐ tagonize Ach and glutamate ionotropic receptors. The neuroprotective activity of PhTx-343 was tested in cerebellar granule cells culture challenged with NMDA and kainate toxicity and compared to that of Arg-636 [109]. Data showed that both polyamines protected cul‐ tures against damage, but Arg-636 was found to be less potent than PhTx-343 against kai‐ nate-induced damage. The structural change in PhTx-343 increased its potency, but in

Due to their lack of selectivity, the use of philanthotoxins as pharmaceuticals may have been limited, and so many modified synthetic analogues were designed for medical treatment

or Ca2+ permeable pore, membrane

http://dx.doi.org/10.5772/ 52382

101

the peptide SNX-482 that blocks R-type voltage dependent calcium channels [103].

lular environments, lead to opening of ASICs Na+

higher doses, toxic side effects, were observed.

depolarization and increase in Ca2+ intracellular concentration [104].

lying neuroprotection as well as the design of novel neuroprotective agents.

prevent dopamine neurons cell death, possibly by the inhibition of Jun activation.

The fraction of the venom of the spider *Agelenopsis aperta* containing argiotoxin, was first demonstrated to have anticonvulsant in NMDA-induced and audiogenic seizures [91]. The synthetic analogue of argiotoxin, Arg-636, is a selective antagonist of NMDA receptors bind‐ ing to the Mg2+ binding site at the receptor with anticonvulsant and neuroprotective actions. In addition, from the venom of *A. aperta,* another NMDA receptor blocker, Agatoxin 489 was reported as anticonvulsant against kainate-induced seizures and its synthetic analogue Agel-505, was able to block cationic currents in oocytes transfected with NMDA receptor cDNA [92].

Aside from antagonizing glutamate receptors, arthropod neurotoxins may exert anticonvul‐ sant and neuroprotective effects targeting other neurotransmitter systems. The venom of the Brazilian spider *Phoneutria nigriventer* has been extensively studied over the past 20 years. Neurotoxins isolated from the venom of *P. nigriventer*, such as PhTx-3 (Tx-3) were reported to inhibit Ca2+ dependent-glutamate release [47]. Tx3-3 and Tx3-4 also inhibit voltage-acti‐ vated Ca2+ channels of P/Q type [93] and recently their neuroprotective activity was tested. According to [94], Tx3-3 and Tx3-4 protected hippocampal slices against damage and cell death induced by ischemic insult resulted from low oxygen and low glucose. Moreover, PhTx3, Tx3-3, and Tx3-4, inhibited cell loss in retinal slices submitted to the same ischemic protocol [95]. Another Brazilian species lives in Cerrado, the colonial spider *Parawixia bistria‐ ta* and has many neuroactive molecules with different modes of action [96]. Parawixin-1 was the first isolated neurotoxin from *P. bistriata* venom. In experiments using rat retinas, sub‐ mitted to ischemic insult, the intravitreal injection of Parawixin inhibited cell loss [97], prob‐ ably through a potent and specific enhancing action on glutamate transporters type EAAT2 [98]. Another neurotoxin isolated from the venom of *P. bistriata*, Parawixin II, formerly, FrPbAII, inhibited GABA and glycine uptakes in synaptosomes from rat cerebral cortices. In addition, the administration of Parawixin II into the vitreous humor of Wistar rats protected retinal neurons against ischemic insult resulted from an increase in the intra ocular pressure [96]. Data also show that Parawixin II blocked seizures induced by the injection of GABAer‐ gic antagonists, bicuculline [99], pentylenetetrazole (PTZ) and picrotoxin, as well as pilocar‐ pine and kainic acid [100]. It is worth noting that the acute injection of Parawixin II does not alter rat behavior in the open field and repeated central injection does not impair acquisition and learning in the Morris water maze. Finally, Parawixin II induces ataxia in the rotarod in doses far higher than effective doses, indicating good therapeutic indexes [100].

Also from South America, the Chilean giant pink tarantula *Grammostola spatulata* paralyzes its preys by injecting a mixture of toxins that blocks ion channels [101]. w-Grammotoxin SIA was isolated from the venom of *G. spatulata* and the potent blocking effect over N-, P-, and Q-type but not L-type of voltage gated calcium channels was reported [102]. The antagonis‐ tic activity of w-grammotoxin over voltage dependent calcium channels is considered a therapeutic option to be used in neurodegenerative disorders such as ischemia.

The African tarantula *Hysterocrates gigas* known as the giant baboon spider, inhabits the rain forests of West Africa. The isolation of the venom of *H. gigas*, resulted in the identification of the peptide SNX-482 that blocks R-type voltage dependent calcium channels [103].

PA/kainate glutamate receptors make mutant neurons more vulnerable to damage and

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The fraction of the venom of the spider *Agelenopsis aperta* containing argiotoxin, was first demonstrated to have anticonvulsant in NMDA-induced and audiogenic seizures [91]. The synthetic analogue of argiotoxin, Arg-636, is a selective antagonist of NMDA receptors bind‐ ing to the Mg2+ binding site at the receptor with anticonvulsant and neuroprotective actions. In addition, from the venom of *A. aperta,* another NMDA receptor blocker, Agatoxin 489 was reported as anticonvulsant against kainate-induced seizures and its synthetic analogue Agel-505, was able to block cationic currents in oocytes transfected with NMDA receptor

Aside from antagonizing glutamate receptors, arthropod neurotoxins may exert anticonvul‐ sant and neuroprotective effects targeting other neurotransmitter systems. The venom of the Brazilian spider *Phoneutria nigriventer* has been extensively studied over the past 20 years. Neurotoxins isolated from the venom of *P. nigriventer*, such as PhTx-3 (Tx-3) were reported to inhibit Ca2+ dependent-glutamate release [47]. Tx3-3 and Tx3-4 also inhibit voltage-acti‐ vated Ca2+ channels of P/Q type [93] and recently their neuroprotective activity was tested. According to [94], Tx3-3 and Tx3-4 protected hippocampal slices against damage and cell death induced by ischemic insult resulted from low oxygen and low glucose. Moreover, PhTx3, Tx3-3, and Tx3-4, inhibited cell loss in retinal slices submitted to the same ischemic protocol [95]. Another Brazilian species lives in Cerrado, the colonial spider *Parawixia bistria‐ ta* and has many neuroactive molecules with different modes of action [96]. Parawixin-1 was the first isolated neurotoxin from *P. bistriata* venom. In experiments using rat retinas, sub‐ mitted to ischemic insult, the intravitreal injection of Parawixin inhibited cell loss [97], prob‐ ably through a potent and specific enhancing action on glutamate transporters type EAAT2 [98]. Another neurotoxin isolated from the venom of *P. bistriata*, Parawixin II, formerly, FrPbAII, inhibited GABA and glycine uptakes in synaptosomes from rat cerebral cortices. In addition, the administration of Parawixin II into the vitreous humor of Wistar rats protected retinal neurons against ischemic insult resulted from an increase in the intra ocular pressure [96]. Data also show that Parawixin II blocked seizures induced by the injection of GABAer‐ gic antagonists, bicuculline [99], pentylenetetrazole (PTZ) and picrotoxin, as well as pilocar‐ pine and kainic acid [100]. It is worth noting that the acute injection of Parawixin II does not alter rat behavior in the open field and repeated central injection does not impair acquisition and learning in the Morris water maze. Finally, Parawixin II induces ataxia in the rotarod in

doses far higher than effective doses, indicating good therapeutic indexes [100].

therapeutic option to be used in neurodegenerative disorders such as ischemia.

Also from South America, the Chilean giant pink tarantula *Grammostola spatulata* paralyzes its preys by injecting a mixture of toxins that blocks ion channels [101]. w-Grammotoxin SIA was isolated from the venom of *G. spatulata* and the potent blocking effect over N-, P-, and Q-type but not L-type of voltage gated calcium channels was reported [102]. The antagonis‐ tic activity of w-grammotoxin over voltage dependent calcium channels is considered a

therefore, JSTX-3 is an interesting neuroprotective agent in this model.

cDNA [92].

Applications

100

The arboreal tarantula *Psalmopoeus cambridgei* is an aggressive spider that lives in the tropi‐ cal forests of Trindad. As mentioned before, PcTx-1 (π-theraphotoxin-Pc1a) present in *P. cambridgei* venom is the only gating modifier of ASICs [50]. In addition to pain inhibitor, it exerts an interesting neuroprotective and a possible antidepressant activity due to the in‐ volvement of ASICs in cell excitability. A drop in pH from neutral 7.4 to more acid extracel‐ lular environments, lead to opening of ASICs Na+ or Ca2+ permeable pore, membrane depolarization and increase in Ca2+ intracellular concentration [104].

In the light of these facts, Yang and coworkers [105] investigated the neuroprotective activi‐ ty of PcTx in neurons from newborn piglets submitted to a model of asphyxia-induced car‐ diac arrest. Data show that the administration of PcTx before the hypoxia-ischemia insult partially prevents the death of neurons in putamen, the most vulnerable encephalic area in this model. The addition of MK-801, a NMDA antagonist, in combination with PcTx exerted better results in cell survival, but in low doses of MK-801. In addition to protection of neuro‐ nal cells, treatment with PcTx accelerated neurologic recovery. These results point PcTx as a very unique neurotoxin that should be used as tool in the investigation of processes under‐ lying neuroprotection as well as the design of novel neuroprotective agents.

Bees and wasps are part of the group of the insects, whose stings release a cocktail of toxins, including enzymes, peptides and biogenic amines [106]. Toxins in bee venom have received attention for their properties as anti-inflammatory agents, and in many countries, physicians even prescribe bee stings as treatment of rheumatologic diseases. Recently, Doo and collea‐ gues [107] showed that the bee venom when injected in rats with induced Parkinson disease prevent dopamine neurons cell death, possibly by the inhibition of Jun activation.

Regarding solitary wasps, the most studied wasp species is the European beewolf, *Philan‐ thus triangulum*, the natural predators of honeybees. The adult individuals of this species are herbivores, whereas the larvae eat the paralyzed bees brought to the colony by foraging wasps. The isolation of venom contents begun in the early 80s and revealed that among oth‐ er classes of molecules, *P. triangulum* venom contains potent acylpolyamines [86]. Philantho‐ toxins, like other acylpolyamines are mostly potent and selective antagonists of vertebrate and invertebrate glutamate receptors, particularly AMPA receptors [108]. The first isolated and most studied philathotoxin is PhTX-433 and its synthetic analogue, PhTx-343, which an‐ tagonize Ach and glutamate ionotropic receptors. The neuroprotective activity of PhTx-343 was tested in cerebellar granule cells culture challenged with NMDA and kainate toxicity and compared to that of Arg-636 [109]. Data showed that both polyamines protected cul‐ tures against damage, but Arg-636 was found to be less potent than PhTx-343 against kai‐ nate-induced damage. The structural change in PhTx-343 increased its potency, but in higher doses, toxic side effects, were observed.

Due to their lack of selectivity, the use of philanthotoxins as pharmaceuticals may have been limited, and so many modified synthetic analogues were designed for medical treatment

purposes, so far [82]. However, the use of philanthotoxins and other polyamines as tools in research investigation has aided the understanding of several synaptic mechanisms. As it has been recently shown using Ca2+-permeable AMPA receptors expressed in HEK cells. Ac‐ cording to [110] the block of these AMPA receptors by PhTx-74, a synthetic analogue of PhTX-433 will reflect structural and biophysical parameters of the channel, such as its subu‐ nit composition and mean conductance, respectively. In addition, the investigation of the antagonistic activity of PhTx-343 over ACh receptors showed that the interaction of the tox‐ in with nicotinic receptors is largely voltage dependent, slow and uncompetitive, a similar mode by which they block glutamate ionotropic channels [111].

**5. Actions on mood disorders**

both, in basic and clinical science.

tribute for the development of novel drugs.

According to the World Health Organization, depression, one of the most important mood disorders, affects up to 5-10% of people worldwide at any time of their lives. Patients with a diagnosed mood disorder are more likely to be women, in productive years, 20 to 40 yearold, and will need in most cases, psychotherapy and/or pharmacological intervention. The costs of these psychiatric and/or psychological disorders are immense, since they affect peo‐ ple regardless of education or socioeconomic status, accounting in the worse cases, for a huge number of suicides. In the United States up to 95% of all suicides, involve mentally ill people, accounting for 1.3% of all deaths [118]. A recent survey shows that generalized anxi‐ ety disorder, posttraumatic stress disorder, social anxiety disorder and panic disorder are highly predictive of suicidal idealization [118]. Many aspects of the pathophysiology of mood disorders, as well as the regulation of normal mood states remain unknown. Howev‐ er, with the improvement of techniques for research and diagnosis, such as positron emis‐ sion tomography, magnetic resonance and multiple channels recording electroencephalogram, soon researchers will be capable to identify structures and neuro‐ chemical mechanism involved in the regulation of mental states, including mood. So far, we know that limbic structures, such as the amygdala, hippocampus, hypothalamus and prefrontal cortex control the emotional aspects of brain function. There are plenty of connec‐ tions among these structures, which might be involved in the onset of mood disorders [119]. Pharmacological treatment of mood disorders consists in daily intake of anti-depressants, anxiolytics or anti-psychotics, most of which cause a wide set of undesired side effects that impose restrictions to patients quality of life. In this regard novel drugs prescribed for mood disorder, such as serotonin uptake inhibitors might be better tolerated and safer than classi‐ cal drugs, such as monoamine oxidase inhibitors. Among the observed undesired effects we can cite; dizziness, sedation, sexual dysfunction and suicidal though, a paradoxical effect of serotonin uptake inhibitors [119]. Aside from tolerability, medicines used as treatment of de‐ pression for example, take too long to produce effect in only a minority of patients; 35-45% of treated patients [120]. Therefore, there is a still great need for novel alternatives to be used

New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

http://dx.doi.org/10.5772/ 52382

103

The neurochemistry of mood disorders is complex and there is a list of candidates for tar‐ gets of mood stabilizers, such as adrenaline, GABA, serotonin and glutamate receptors and transporters. There is not many works relating neurotoxins from arthropods and mood dis‐ orders, but the few works available showed that in some cases, these molecules might con‐

The venom of the Brazilian colonial spider *Parawixia bistriata* was fractionated and tested in many animal models of epilepsy, neurodegeneration and anxiety. According to [121], the microinjection of Parawixin 2 (formerly FrPbAII) in the dorsal hippocampus of male Wistar rats increased the time spent in the open arms of the elevated plus maze. Moreover, rats ex‐ posed to the light-dark choice apparatus spent more time in the light side of the box, similar‐ ly to what observed for diazepam or nipecotic acid, a GABA transporter-1 (GAT-1) inhibitor [121]. In another investigation of *P. bistriata* venom contents, Saidemberg and co-workers

Going further on wasp venoms, the anticonvulsant and/or neuroprotective effects of mole‐ cules in the venom of two Brazilian social species of the genus *Polybia*, were investigated. According to Cunha and co-workers [112] and Mortari and co-workers [113], the non-enzy‐ matic fraction of the venoms of *Polybia ignobilis* and *Polybia occidentalis* inhibit seizures evoked by the injection of several chemoconvulsants in Wistar rats. The neuroactive mole‐ cules present in the venom of *P. ignobilis* and *P. occidentalis* are now in phase of structurefunction investigation.

Finally, neurotoxins from scorpion venoms have been subject of a wide range of works, mostly approaching the identification of voltage-dependent ion channel activators/blockers. The neuroprotective and/or anticonvulsant activity of these peptides, in turn have received a few lines of investigation [3] despite the ancient use of these animals whole or parts, in the popular medicine in oriental countries, like China [20]. One of the most studied species is the Asian scorpio*n Buthus martensi Karsch* whose venom has several neuroactive peptides, among whose, we may find BmK AEP, which was the first anticonvulsant peptide isolated from scorpion venoms. According to [28], the injection of BmK AEP blocked seizures in‐ duced by the injection of coriaria lactone in doses causing no visible side effects [114]. Fur‐ ther isolation of venom of *B. martensi* led to the identification of other peptides, such as BmK AS and BmK Ts and other mostly with analgesic activity. According to Zhao and co-workers [115] BmK AS, a sodium channel modulator at site-4 receptor, inhibited PTZ induced behav‐ ioral and electroencephalographic seizures and decreased mean score of pilocarpine-in‐ duced seizures. Moreover, these authors showed that BmK AS does not impair locomotion or motor behavior.

The venom of the Mexican scorpion *Centruroides limpidus limpidus*, was fractionated and many activators of voltage-gated ion channel ligands were identified [116]. An exception is Cll9, which stands for *Centruroides limpidus limpidus* toxin nr 9. Cll9 is a 63-residue peptide that has a divergent mode of action; it inhibits sodium channels in superior cervical gan‐ glion neurons and [117]. When injected in Wistar rats via i.c.v., Cll9 inhibited behavioral and electroencephalographic seizures evoked by the microinjection of penicillin into the basolat‐ eral amygdala. It is worth noticing that Cll9 has no effect on arthropods such as crickets or crayfish like many sodium channels modulators found in scorpion venoms.
