Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

Combined Peptidomic and Proteomic Analysis of Electrically Stimulated and Manually Dissected Venom from the South American Bullet Ant Paraponera clavata.

Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with <20% shared by the two collection methods. There was also a marked difference between venoms obtained by venom gland dissection from different ant colonies. These findings demonstrate the rich composition and variability of P. clavata venom.

Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms.

Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.

Nerve-muscle activation by rotating permanent magnet configurations.

KEY POINTS: The standard method of magnetic nerve activation using pulses of high current in coils has drawbacks of high cost, high electrical power (of order 1 kW), and limited repetition rate without liquid cooling. Here we report a new technique for nerve activation using high speed rotation of permanent magnet configurations, generating a sustained sinusoidal electric field using very low power (of order 10 W). A high ratio of the electric field gradient divided by frequency is shown to be the key indicator for nerve activation at high frequencies. Activation of the cane toad sciatic nerve and attached gastrocnemius muscle was observed at frequencies as low as 180 Hz for activation of the muscle directly and 230 Hz for curved nerves, but probably not in straight sections of nerve. These results, employing the first prototype device, suggest the opportunity for a new class of small low-cost magnetic nerve and/or muscle stimulators. ABSTRACT: Conventional pulsed current systems for magnetic neurostimulation are large and expensive and have limited repetition rate because of overheating. Here we report a new technique for nerve activation, namely high-speed rotation of a configuration of permanent magnets. Analytical solutions of the cable equation are derived for the oscillating electric field generated, which has amplitude proportional to the rotation speed. The prototype device built comprised a configuration of two cylindrical magnets with antiparallel magnetisations, made to rotate by interaction between the magnets' own magnetic field and three-phase currents in coils mounted on one side of the device. The electric field in a rectangular bath placed on top of the device was both numerically evaluated and measured. The ratio of the electric field gradient on frequency was approximately 1 V m(-2) Hz(-1) near the device. An exploratory series of physiological tests was conducted on the sciatic nerve and attached gastrocnemius muscle of the cane toad (Bufo marinus). Activation was readily observed of the muscle directly, at frequencies as low as 180 Hz, and of nerves bent around insulators, at frequencies as low as 230 Hz. Nerve-muscles, with the muscle elevated to avoid its direct activation, were occasionally activated, possibly in the straight section of the nerve, but more likely in the nerve where it curved up to the muscle, at radius of curvature 10 mm or more, or at the nerve end. These positive first results suggest the opportunity for a new class of small, low-cost devices for magnetic stimulation of nerves and/or muscles.

The complexity and structural diversity of ant venom peptidomes is revealed by mass spectrometry profiling.

RATIONALE: Compared with other animal venoms, ant venoms remain little explored. Ants have evolved complex venoms to rapidly immobilize arthropod prey and to protect their colonies from predators and pathogens. Many ants have retained peptide-rich venoms that are similar to those of other arthropod groups. METHODS: With the goal of conducting a broad and comprehensive survey of ant venom peptide diversity, we investigated the peptide composition of venoms from 82 stinging ant species from nine subfamilies using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). We also conducted an in-depth investigation of eight venoms using reversed-phase high-performance liquid chromatography (RP-HPLC) separation coupled with offline MALDI-TOFMS. RESULTS: Our results reveal that the peptide compositions of ant venom peptidomes from both poneroid and formicoid ant clades comprise hundreds of small peptides (<4 kDa), while large peptides (>4 kDa) are also present in the venom of formicoids. Chemical reduction revealed the presence of disulfide-linked peptides in most ant subfamilies, including peptides structured by one, two or three disulfide bonds as well as dimeric peptides reticulated by three disulfide bonds. CONCLUSIONS: The biochemical complexity of ant venoms, associated with an enormous ecological and taxonomic diversity, suggests that stinging ant venoms constitute a promising source of bioactive molecules that could be exploited in the search for novel drug and biopesticide leads.

Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif.

The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed ß-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U(1)-liotoxin-Lw1a (U(1)-LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first example of a native peptide that adopts the DDH fold. U(1)-LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U(1)-LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development.

ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures.

ArachnoServer (www.arachnoserver.org) is a manually curated database providing information on the sequence, structure and biological activity of protein toxins from spider venoms. These proteins are of interest to a wide range of biologists due to their diverse applications in medicine, neuroscience, pharmacology, drug discovery and agriculture. ArachnoServer currently manages 1078 protein sequences, 759 nucleic acid sequences and 56 protein structures. Key features of ArachnoServer include a molecular target ontology designed specifically for venom toxins, current and historic taxonomic information and a powerful advanced search interface. The following significant improvements have been implemented in version 2.0: (i) the average and monoisotopic molecular masses of both the reduced and oxidized form of each mature toxin are provided; (ii) the advanced search feature now enables searches on the basis of toxin mass, external database accession numbers and publication date in ArachnoServer; (iii) toxins can now be browsed on the basis of their phyletic specificity; (iv) rapid BLAST searches based on the mature toxin sequence can be performed directly from the toxin card; (v) private silos can be requested from research groups engaged in venoms-based research, enabling them to easily manage and securely store data during the process of toxin discovery; and (vi) a detailed user manual is now available.

A novel family of insect-selective peptide neurotoxins targeting insect large-conductance calcium-activated K+ channels isolated from the venom of the theraphosid spider Eucratoscelus constrictus.

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (κ-TRTX-Ec2a, κ-TRTX-Ec2b, and κ-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that κ-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K(+) (BK(Ca)) channel currents with IC(50) values of 3 to 25 nM. In addition, κ-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K(+) currents, but only at significantly higher concentrations. κ-TRTX-Ec2a and κ-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous κ-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, κ-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, κ-TRTX-Ec2a and κ-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK(Ca) channels. This study therefore establishes these toxins as tools for studying the role of BK(Ca) channels in insects and lead compounds for the development of novel insecticides.

Label-Free, Real-Time Phospholipase-A Isoform Assay.

Phospholipase-A (PLA) enzymes catalyze the hydrolysis of ester bonds in select glycerophospholipids. Sensors for rapidly measuring the PLA activity in biological samples have relevance in the study of venom compositions and in medical diagnostics for the diagnosis of diseases such as acute pancreatitis. Current PLA sensor technologies are often restricted by the time it takes to prepare an assay, the necessity of using fluorescent labels, or the fact they might require strict pH control of the buffer vehicles used. Here we present a tethered bilayer lipid membrane (tBLM) impedance sensor array for the rapid and real-time detection of PLA, which includes the ability to selectively detect phospholipase-A2 (PLA2) from phospholipase-A1 (PLA1) isoforms. Comparing the activity of PLA1 and PLA2 in an array of tBLMs composed of ether phospholipids, ester phospholipids or ether-ester phospholipids allows for the rapid and reliable distinction between the isoforms, as measured using swept-frequency electrical impedance spectroscopy. After testing the assay using pure enzymes, we demonstrate the capacity of the sensor to identify specific PLA2-type, calcium-dependent activity from the venom of the South American bullet ant, Paraponera clavata, at a concentration of 1 µg/mL. The specificity of the phospholipase activity was corroborated using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. As further validation, we tested the activities of a PLA1 isoform in the presence of different buffers commonly used in biology and biochemistry experiments. Sensitivity testing shows that PLA1 can be detected at an activity as low as 0.06 U/mL. The rapid and reliable detection of phospholipases presented in this study has potential applications in the study of animal venoms as well as in lipase bioreactors and point-of-care devices.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

The Janus-faced atracotoxins are specific blockers of invertebrate K(Ca) channels.

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.

A rational nomenclature for naming peptide toxins from spiders and other venomous animals.

Molecular toxinology research was initially driven by an interest in the small subset of animal toxins that are lethal to humans. However, the realization that many venomous creatures possess a complex repertoire of bioactive peptide toxins with potential pharmaceutical and agrochemical applications has led to an explosion in the number of new peptide toxins being discovered and characterized. Unfortunately, this increased awareness of peptide-toxin diversity has not been matched by the development of a generic nomenclature that enables these toxins to be rationally classified, catalogued, and compared. In this article, we introduce a rational nomenclature that can be applied to the naming of peptide toxins from spiders and other venomous animals.

Intersexual variations in Northern (Missulena pruinosa) and Eastern (M. bradleyi) mouse spider venom.

Venoms of both sexes of Australian Northern (Missulena pruinosa) and Eastern (Missulena bradleyi) mouse spiders were studied in order to determine intersexual variations in venom yield, composition and bioactivity. Females of both species yielded more venom than males. High-performance liquid chromatography (HPLC) and mass spectrometry data further indicate a substantial degree of intersexual variation in the venom composition of both species. In a cricket (Acheta domestica) acute toxicity assay, only small intersexual differences were observed, but M. bradleyi venom was found to be considerably more potent than M. pruinosa venom. In the chick biventer cervicis nerve-muscle preparation, male but not female M. bradleyi venom induced large and sustained muscle contractions with fasciculation and decreased twitch height that could be reversed by CSL funnel-web spider antivenom. In contrast, venoms of both sexes of M. pruinosa did not induce significant effects in the chick biventer cervicis nerve-muscle preparation. We therefore conclude that female M. bradleyi venom and venoms from male and female M. pruinosa appear to contain few, if any, orthologs of delta-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These findings are consistent with clinical reports that mouse spiders, particularly species other than male M. bradleyi, do not appear to be a major medical problem in humans.

Evaluation of Chemical Strategies for Improving the Stability and Oral Toxicity of Insecticidal Peptides.

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons.

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

The omega-atracotoxins: selective blockers of insect M-LVA and HVA calcium channels.

The omega-atracotoxins (omega-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, omega-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the omega-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 microM. To further characterize the target of the omega-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that omega-ACTX-Ar1a, and its homolog omega-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca(v)) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca(v) channel activation, and was voltage-independent, suggesting that omega-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 microM omega-ACTX-Ar1a failed to significantly affect global K(v) channel currents. However, 1 microM omega-ACTX-Ar1a caused a modest 18% block of insect Na(v) channel currents, similar to the minor block of Na(v) channels reported for other insect Ca(v) channel blockers such as omega-agatoxin IVA. These findings validate both M-LVA and HVA Ca(v) channels as potential targets for insecticides.

Fighting the global pest problem: preface to the special Toxicon issue on insecticidal toxins and their potential for insect pest control.

Arthropod pests are responsible for major crop devastation and are vectors for the transmission of new and re-emerging diseases in humans and livestock. Despite many years of effective control by conventional agrochemical insecticides, a number of factors are threatening the effectiveness and continued use of these agents. These include the development of insecticide resistance and use-cancellation or de-registration of some insecticides due to human health and environmental concerns. Several approaches are being investigated for the design of new (bio)pesticides. These include the development of transgenic plants and recombinant baculoviruses as delivery systems for a variety of insect-selective toxins. Additional approaches for the development of foliar sprays include the rational design of peptidomimetics based on the key residues of these toxins that interact with the insect target. This special issue provides an overview of these phyletically selective animal, plant and microbial toxins and their diverse mechanisms of action to paralyze or kill arthropods. In addition, it reviews their potential for biopesticide discovery and validation of novel insecticide targets and provides an overview of the strengths and weaknesses of biopesticides in the global control of arthropod pests.

Insect-selective spider toxins targeting voltage-gated sodium channels.

The voltage-gated sodium (Na(v)) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Na(v) channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of these sites. These all conform to an 'inhibitor cystine-knot' motif with structural, but not sequence homology, to a variety of other spider and marine snail toxins. Of these, spider toxins several show phyla-specificity and are being considered as lead compounds for the development of biopesticides. Hainantoxin-I appears to target site-1 to block Na(v) channel conductance. Magi 2 and Tx4(6-1) slow Na(v) channel inactivation via an interaction with site-3. The delta-palutoxins, and most likely mu-agatoxins and curtatoxins, target site-4. However, their action is complex with the mu-agatoxins causing a hyperpolarizing shift in the voltage-dependence of activation, an action analogous to scorpion beta-toxins, but with both delta-palutoxins and mu-agatoxins slowing Na(v) channel inactivation, a site-3-like action. In addition, several other spider neurotoxins, such as delta-atracotoxins, are known to target both insect and vertebrate Na(v) channels most likely as a result of the conserved structures within domains of voltage-gated ion channels across phyla. These toxins may provide tools to establish the molecular determinants of invertebrate selectivity. These studies are being greatly assisted by the determination of the pharmacophore of these toxins, but without precise identification of their binding site and mode of action their potential in the above areas remains underdeveloped.