**4. Biotechnological and pharmacological applications of spider venom toxins**

Spider venoms contain a complex mixture of proteins, polypeptides, neurotoxins, nucleic acids, free amino acids, inorganic salts and monoamines that cause diverse effects in verte‐ brates and invertebrates [145]. Regarding the pharmacology and biochemistry of spider ven‐ oms, they present a variety of ion channel toxins, novel non-neurotoxins, enzymes and low molecular weight compounds [146].

### **4.1. Toxins acting on cardiovascular system**

Venom from the South American tarantula *Grammostola spatulata* presents GsMtx-4, a small peptide belonging to the "cysteine-knot" family that blocks cardiac stretch-activated ion channels and suppresses atrial fibrillation in rabbits [147]. Studies are being conducted to develop therapeutics for atrial fibrillation based on GsMtx-4.

### **4.2. Toxins acting on hemostasis**

ARACHnase (Hemostasis Diagnostics International Co., Denver, CO) is a normal plasma that contains a venom extract from the brown recluse spider, *Loxosceles reclusa*, which mimics the presence of a lupus anticoagulant (LA). ARACHnase is a biotechnological product useful‐ ness like a positive control for lupus anticoagulant testing [148]. Native dermonecrotic tox‐ ins (phospholipase-D) from *Loxosceles* sp. are agents that stimulate platelet aggregation [149].

### **4.3. Toxins with antibiotic activity**

was observed in the presence of Bjα IT [136] and OD1 [137], which are toxins from *Buthotus judaicus* and *Odonthobuthus doriae* scorpion venom, respectively. A second group of scorpion toxins slowing insect sodium channel inactivation was called alpha-like toxins. The first pre‐ cisely described toxins from this group were the Lqh III/Lqh3 (from *L. q. hebraeus*), Bom III/ Bom 3 and Bom IV/ Bom 4 (from *B. o. mardochei*). They were all tested on cockroach axonal preparation [138-139]. BmKM1 toxin from *B. martensi* Karsch was the first alpha-like toxin available in recombinant form that was tested also on cockroach axonal preparation [140]. Toxins Lqh6 and Lqh7 from *L. q. hebraeus* scorpion venom show high structural similarity with Lqh3 toxin. Their toxicity to cockroach is in the range found for other alpha-like toxins [141]. Alpha-like toxins from scorpion venoms show lower efficiency when applied to in‐ sects, as compared to α anti-insect toxins. Therefore they seem to be less interesting from the point of view of future insecticide development [132]. Scorpion contractive and depressant toxins are highly selective for insect sodium channels. Several of these toxins were tested on cockroach axonal preparations; toxin AaH IT1 from the *A. australis* scorpion venom was the first one [142-143]. All other contractive toxins tested on cockroach axon produced very sim‐ ilar effects, as for example Lqq IT1 from *L. q. quinquestriatus* [133]; Bj IT1 from *B. judaicus*

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

**4. Biotechnological and pharmacological applications of spider venom**

Spider venoms contain a complex mixture of proteins, polypeptides, neurotoxins, nucleic acids, free amino acids, inorganic salts and monoamines that cause diverse effects in verte‐ brates and invertebrates [145]. Regarding the pharmacology and biochemistry of spider ven‐ oms, they present a variety of ion channel toxins, novel non-neurotoxins, enzymes and low

Venom from the South American tarantula *Grammostola spatulata* presents GsMtx-4, a small peptide belonging to the "cysteine-knot" family that blocks cardiac stretch-activated ion channels and suppresses atrial fibrillation in rabbits [147]. Studies are being conducted to

ARACHnase (Hemostasis Diagnostics International Co., Denver, CO) is a normal plasma that contains a venom extract from the brown recluse spider, *Loxosceles reclusa*, which mimics the presence of a lupus anticoagulant (LA). ARACHnase is a biotechnological product useful‐ ness like a positive control for lupus anticoagulant testing [148]. Native dermonecrotic tox‐ ins (phospholipase-D) from *Loxosceles* sp. are agents that stimulate platelet aggregation [149].

[143], Bm 32-1 and Bm 33-1 from *B. martensi* [144].

molecular weight compounds [146].

**4.2. Toxins acting on hemostasis**

**4.1. Toxins acting on cardiovascular system**

develop therapeutics for atrial fibrillation based on GsMtx-4.

**toxins**

Applications

32

Two peptide toxins with antimicrobial activity, lycotoxins I and II, were identified from ven‐ om of the wolf spider *Lycosa carolinensis* (Araneae: Lycosidae). The lycotoxins may play a dual role in spider-prey interaction, functioning both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion. Spi‐ der venoms may represent a potentially new source of novel antimicrobial agents with im‐ portant medical implications [150].

#### **4.4. Toxins acting on inflammatory and nociceptive response**

Psalmotoxin 1, a peptide extracted from the South American tarantula *Psalmopoeus cambridg‐ ei*, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway [151]. Phospholipases from both *Loxosceles laeta* and *Loxosceles reclusa* cleaved LPC (lysophosphati‐ dylcholine) to LPA (lysophosphatidic acid) and choline. LPA receptors are potential targets for *Loxosceles* sp. envenomation treatment [152]. The possibilities for biotechnological appli‐ cations in this area are enormous. Recombinant dermonecrotic toxins could be used as re‐ agents to establish a new model to study the inflammatory response, as positive inducers of the inflammatory response and edema [9, 153-154]. The phospholipase-D from *Loxosceles* venom could be used in phospholipid studies, specially studies on cell membrane constitu‐ ents with emphasis upon sphingophospholipids, lysophospholipids, lysophosphatidic acid and ceramide-1-phosphate, as models for elucidating lipid product receptors, signaling pathways and biological activities; this new wide field of *Loxosceles* research could also re‐ veal new targets for the treatment of envenomation [10].

#### **4.5. Toxins acting on immunological system**

The antiserum most commonly used for treatment of loxoscelism in Brazil is anti-arachnidic serum. This serum is produced by the Instituto Butantan (São Paulo, Brazil) by hyperimmu‐ nization of horses with venoms of the spiders *Loxosceles gaucho* and *Phoneutria nigriventer* and the scorpion *Tityus serrulatus*. Several studies have indicated that sphingomyelinase D (SMase D) in venom of *Loxosceles* sp. spiders is the main component responsible for local and systemic effects observed in loxoscelism [153, 155]. Neutralization tests showed that an‐ ti-SMase D serum has a higher activity against toxic effects of *L. intermedia* and *L. laeta* ven‐ oms and similar or slightly weaker activity against toxic biological effects of *L. gaucho* than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy [155].

#### **4.6. Toxins with anticancer and cytotoxic activities**

Psalmotoxin 1 was evaluated on inhibited Na+ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). These observations suggest this toxin may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel

type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas [156]. The antitumor activity of a potent antimicrobial peptide isolated from hemocytes of the spider *Acanthoscurria gomesiana*, named gomesin, was tested *in vitro* and *in vivo*. Gomesin showed cytotoxic and antitumor activities in cell lines, such as melano‐ ma, breast cancer and colon carcinoma [157].

cous slime are secreted permanently, containing substances with different pharmacologic activities such as cardiotoxins, neurotoxins, hypotensive as well as hypertensive agents, he‐ molysins, and many others. Chemically they belong to a wide variety of substance classes such as steroids, alkaloids, indolalkylamines, catecholamines and low molecular peptides [11, 163]. Several studies have been showing new potential molecules for a variety of phar‐

Toxins from Venomous Animals: Gene Cloning, Protein Expression and Biotechnological Applications

Neurotensin-like peptides has been identified from frog skin, such as margaratensin, isolat‐ ed from *Rana margaratae* [164], a potential antihypertensive drug. Similar to the cardiac gly‐ cosides, bufadienolides from *Bufo bufo gargarizans* toad skin are able of inhibiting Na+

ATPase, having an important role on treatment of congestive heart failure and arterial hypertension [165]. Examples of these bufadienolides are arenobufagin [166], cinobufagin, bufalin, resibufogenin, among others [165]. In the skin of *Rana temporaria* and *Rana igromacu‐ lata* frogs, bradykinin, a hypotensive and smooth muscle exciting substance, has been found [11]. Atelopidtoxin, a water-soluble toxin from skin of *Atelopus zeteki* frog, when injected into mammals, produces hypotension and ventricular fibrillation [167]. Semi-purified skin ex‐ tracts from *Pseudophryne coriacea* frog displayed effects on systemic blood pressure, reducing

Annexins are a well-known multigene family of Ca2+-regulated membrane-binding and phospholipid-binding proteins. A novel annexin A2 (Bm-ANXA2) was isolated and purified from *Bombina maxima* skin homogenate, being the first annexin A2 protein reported to pos‐

Toxins with antibiotic activity are the most well studied toxins in toads and frogs. Two anti‐ microbial bufadienolides, telocinobufagin and marinobufagin, were isolated from skin se‐ cretions of the Brazilian toad *Bufo rubescens* [170]. Antimicrobial peptides, named syphaxins (SPXs), were isolated from skin secretions of *Leptodactylus syphax* frog [171]. The alkaloids apinaceamine, 6-methyl-spinaceamine isolated from the skin gland secretions of *Leptodacty‐ lus pentadactylus* showed in screening tests bactericidal activity [172]. The cinobufacini and its active components bufalin and cinobufagin, from *Bufo bufo gargarizans* Cantor skin, pre‐ sented anti-hepatitis B virus (HBV) activity [173]. Telocinobufagin from *Rhinella jimi* toad were demonstrated to be active against *Leishmania chagasi* promastigotes and *Trypanosoma cruzi* trypomastigotes, while hellebrigenin, from same source, was active against only *T. cru‐*

/K+ - 35

http://dx.doi.org/10.5772/52380

macological applications from toads and frogs venoms.

**5.1. Toxins acting on cardiovascular system**

it by a probably cholinergic mechanism [168].

sess platelet aggregation-inhibiting activity [169].

**5.2. Toxins acting on hemostasis**

**5.3. Toxins with antibiotic activity**

*zi* trypomastigotes [174].

### **4.7. Toxins with insecticides applications**

Several spider toxins have been studied as potential insecticidal bioactive with great biotech‐ nological possible applications [10]. A component of the venom of the Australian funnel web spider *Hadronyche versuta* that is a calcium channel antagonist retains its biological activity when expressed in a heterologous system. Transgenic expression of this toxin in tobacco effectively protected the plants from *Helicoverpa armigera* and *Spodoptera littoralis* larvae, with 100% mortality within 48h [158]. LiTxx1, LiTxx2 and LiTxx3 from *Loxosceles intermedia* venom were identified containing peptides that were active against *Spodoptera frugiperda*. These venom-derived products open a source of insecticide toxins that could be used as substitutes for chemical defensives and lead to a decrease in environmental prob‐ lems [159]. An insecticidal peptide referred to as Tx4(6-1) was purified from the venom of the spider *Phoneutria nigriventer* by a combination of gel filtration, reverse-phase fast liq‐ uid chromatography on Pep-RPC, reverse-phase high performance liquid chromatography (HPLC) on Vydac C18 and ion-exchange HPLC. The protein contains 48 amino acids includ‐ ing 10 Cys and 6 Lys. The results showed that Tx4(6-1) has no toxicity for mice, and sug‐ gest that it is a specific anti-insect toxin [160]. SMase D and homologs in the SicTox gene family are the most abundantly expressed toxic protein in venoms of *Loxosceles* and *Sicar‐ ius* spiders (Sicariidae). A recombinant SMase D from *Loxosceles arizonica* was obtained and compared its enzymatic and insecticidal activity to that of crude venom. SMase D and crude venom have comparable and high potency in immobilization assays on crickets. These da‐ ta indicate that SMase D is a potent insecticidal toxin, the role for which it presumably evolved [161]. δ-PaluIT1 and δ-paluIT2 are toxins purified from the venom of the spider *Paracoelotes luctuosus*. Similar in sequence to μ-agatoxins from *Agelenopsis aperta*, their phar‐ macological target is the voltage-gated insect sodium channel, of which they alter the inac‐ tivation properties in a way similar to α-scorpion toxins. Electrophysiological experiments on the cloned insect voltage-gated sodium channel heterologously co-expressed with the tipE subunit in *Xenopus laevis* oocytes, that δ-paluIT1 and δ-paluIT2 procure an increase of Na+ current [162]. Recently, several toxins have been isolated from spiders with potential biotechnological application as insecticide.
