Advances in venomics: Modern separation techniques and mass spectrometry.

Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.

Animal Venoms have Potential to Treat Cancer.

The resistance to chemotherapeutics by the cancerous cells has made its treatment more complicated. Animal venoms have emerged as an alternative strategy for anti-cancer therapeutics. Animal venoms are cocktails of complex bioactive chemicals mainly disulfide-rich proteins and peptides with diverse pharmacological actions. The components of venoms are specific, stable, and potent and have the ability to modify their molecular targets thus making them good therapeutics candidates. The isolation of cancer-specific components from animal venoms is one of the exciting strategies in anti-cancer research. This review highlights the identified venom peptides and proteins from different venomous animals like snakes, scorpions, spiders, bees, wasps, snails, toads, frogs and sea anemones and their anticancer activities including inhibition of proliferation of cancer cells, their invasion, cell cycle arrest, induction of apoptosis and the identification of involved signaling pathways.

Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review.

Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or firing aberrantly in the peripheral or central nervous systems. The treatment guidelines for neuropathic pain include antidepressants, calcium channel α2 delta ligands, topical therapy, and opioids as a second-line option. Pharmacotherapy has not been effective in the treatment of neuropathic pain except in the treatment of trigeminal neuralgia with carbamazepine. The inability to properly treat neuropathic pain causes frustration in both the patients and their treating physicians. Venoms, which are classically believed to be causes of pain and death, have peptide components that have been implicated in pain relief. Although some venoms are efficacious and have shown benefits in patients, their side-effect profile precludes their more widespread use. This review identifies and explores the use of venoms in neuropathic pain relief. This treatment can open doors to potential therapeutic targets. We believe that further research into the mechanisms of action of these receptors as well as their functions in nature will provide alternative therapies as well as a window into how they affect neuropathic pain.

Microbial expression of Exendin-4 analog and its efficacy in mice model.

Exendin-4 is a GLP 1 agonist incretin-mimetic peptide hormone comprising 39 amino acids. Exenatide (Byetta®) is a chemically synthesized version of Exendin-4 with an additional C-terminal amidation. Exenatide acts as a GLP-1 receptor agonist. This paper illustrates the method adopted for cloning, fermentation and purification of recombinant Exendin-4 analog expressed in Escherichia coli. The biologically expressed analog was extensively characterized using different orthogonal methods to confirm their biological activity and physicochemical properties. It was observed that the expressed analog showed comparable functional properties as that of Byetta® irrespective of their modes of development. Further, in vivo efficacy of the recombinant Exendin-4 analog was studied in Oral Glucose Tolerance Test (OGTT) in mice models. Byetta® and Exendin-4 analog treated groups showed comparable glucose lowering activity in the OGTT model.

Insects, arachnids and centipedes venom: A powerful weapon against bacteria. A literature review.

Currently, new antimicrobial molecules extracted or obtained by natural sources, could be a valide alternative to traditional antibiotics. Most of these molecules are represented by antimicrobial peptides (AMPs), which are essential compounds of insect, arachnids and centipedes venom. AMPs, due to their strong effectiveness, low resistance rates and peculiar mode of action, seem to have all the suitable features to be a powerful weapon against several bacteria, especially considering the increasing antibiotic-resistance phenomena. The present literature review focuses on the antibacterial activity of bee, wasp, ant, scorpion, spider and scolopendra crude venom and of their main biological active compounds. After a brief overview of each animal and venom use in folkloristic medicine, this review reports, in a comprehensive table, the results obtained by the most relevant and recent researches carried out on the antibacterial activity of different venom and their AMPs. For each considered study, the table summarizes data concerning minimal inhibitory concentration values, minimal bactericidal concentration values, the methods employed, scientific name and common names and provenience of animal species from which the crude venom and its respective compounds were obtained.

Animal venoms as antimicrobial agents.

Hospitals are breeding grounds for many life-threatening bacteria worldwide. Clinically associated gram-positive bacteria such as Staphylococcus aureus/methicillin-resistant S. aureus and many others increase the risk of severe mortality and morbidity. The failure of antibiotics to kill various pathogens due to bacterial resistance highlights the urgent need to develop novel, potent, and less toxic agents from natural sources against various infectious agents. Currently, several promising classes of natural molecules from snake (terrestrial and sea), scorpion, spider, honey bee and wasp venoms hold promise as rich sources of chemotherapeutics against infectious pathogens. Interestingly, snake venom-derived synthetic peptide/snake cathelicidin not only has potent antimicrobial and wound-repair activity but is highly stable and safe. Such molecules are promising candidates for novel venom-based drugs against S. aureus infections. The structure of animal venom proteins/peptides (cysteine rich) consists of hydrophobic α-helices or ß-sheets that produce lethal pores and membrane-damaging effects on bacteria. All these antimicrobial peptides are under early experimental or pre-clinical stages of development. It is therefore important to employ novel tools for the design and the development of new antibiotics from the untapped animal venoms of snake, scorpion, and spider for treating resistant pathogens. To date, snail venom toxins have shown little antibiotic potency against human pathogens.

[Ants: a chemical library of anticancer molecules]. / Fourmis : une chimiothèque de nouveaux anticancéreux.

Animal venoms are complex mixtures containing simple organic molecules, proteins, peptides, and other bioactive elements with extraordinary biological properties associated with their ability to act on a number of molecular receptors in the process of incapacitating their target organisms. In such a context, arthropod venoms are invaluable sources of bioactive substances, with therapeutic interest but the limited availability of some venom such as those from ants, has restricted the potential that these biomolecules could represent. We investigated for the first time transcriptomic expression from the ant species Tetramorium bicarinatum. Four hundred randomly selected clones from cDNA libraries were sequenced and a total of 374 expressed sequence tags (ESTs) were generated. Based on the results of BLAST searches, these sequences were clustered and assembled into 269 contigs. About 72% (269) of these matched BLASTx hits with an interesting diversity and unusual abundance of cellular transcripts (48%) related to gene and protein expression reflecting the specialization of this tissue. In addition, transcripts encoding transposases were relatively highly expressed (14%). It may be that transposable elements are present and that their presence accounts for some of the variation in venom toxins. About twenty per cent of the ESTs were categorized as putative toxins, the major part represented by allergens (48% of the total venom toxins) such as pilosulin 5, sol i 3 and Myp p I and II. Several contigs encoding enzymes, including zinc-metalloproteases (17%) that are likely involved in the processing and activation of venom proteins/peptides, were also identified from the library. In addition, a number of sequences (8%) had no significant similarity to any known sequence which indicates a potential source of for the discovery of new toxins. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of their products, high-throughput expressed sequence tags were generated using Illumina paired-end sequencing technology. A total of 212 371 758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36 042 contigs for which 27 873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the read mapping toxin class revealed and confirmed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%) followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus). These studies provide a first insight of the gene expression scenario of the venom gland of T. bicarinatum which might contribute to acquiring a more comprehensive view about the origin and functional diversity of venom proteins of this ant. Based on such results, we conducted cytotoxic tests from the crude venom of T.bicarinatum ant and reported toxic effect on tumoral cells lines from one of the fifth of the most frequently occurring cancers with a 3-year survival rate of only 30%. In such a context, new therapeutic strategies are essential and the discovery of new molecules in ant venom could be one possible avenue. Thus our project aims to characterize, from the crude venom of T.bicarinatum, the molecule(s) which have potential anti-cancerous toxicity as well as their mechanisms of action.

Advances in venomics.

The term "venomics" was coined to describe the global study of venom and venom glands, targeting comprehensive characterization of the whole toxin profile of a venomous animal by means of proteomics, transcriptomics, genomics and bioinformatics studies. This integrative approach is supported by the rapid evolution of protein, RNA and DNA sequencing techniques, as well as databases, knowledge-bases and biocomputing algorithms. The aim of this review is to illustrate advances in the field of venomics during the last decade, addressing each step of the procedure, from sample collection to data treatment. A special focus is made on new perspectives for a better understanding of the venomous function and for fostering the discovery of new venom-derived drug candidates.

Separation of mono- and di-PEGylate of exenatide and resolution of positional isomers of mono-PEGylates by preparative ion exchange chromatography.

Exenatide is a synthetic version of the 39-mer peptide of Exendin-4, which is an FDA-approved therapeutic against Type II diabetes mellitus. However, exenatide has a very short in-serum half-life and PEGylation have been performed to improve its in-serum stability. PEGylation often yields multivalent binding to non-specific residues, and the desired species should be carefully separated by chromatographies. In this study, we first devised an aqueous-phase, two-step PEGylation process. This consists of thiolation of Lys 12 and 27 residues followed by attachment of PEG-maleimide (10kD) to thiol groups. This process yields various species: mono-PEGylates with positional isomers, di-PEGylate, and other higher MW substances. A prep-grade cationic exchange chromatography (HiTrap SP) at pH 3.0 partially separated mono- and di-PEGylates based on the molar ratio of conjugated PEG and peptide and thus molecular weight of the conjugates. To further investigate the chromatographic separation of positional isomers of mono-PEGylates, we prepared two kinds of exenatide analogs by point mutation; K12C and K27C. Each analog was mono-PEGylated with very high yield (>95%). When a mixture of the two positional isomers of mono-PEGylates was applied to HiTrap SP chromatography, K12C-PEGylate and K27C-PEGylate eluted separately at 0.22M and 0.33M NaCl, respectively. When the proportions of acid and its conjugate base of the amino acid residues adjacent to the PEGylation site at pH 3.0 were analyzed, K27C-PEGylate shows stronger positive charge than K12C-PEGylate, and we propose the residence time difference between the two mono-PEGylates could be due to the charge difference. ELISA result shows that the immuno-binding activity of both analogs and their mono-PEGylates are well maintained. Furthermore, both mono-PEGylates of the analogs show higher than 50-fold improved anti-trypsin stability. We expect that mono-PEGylates of the exenatide analogs are alternatives to the conventional C40-PEG.

Functional hydrophilic polystyrene beads with uniformly size and high cross-linking degree facilitated rapid separation of exenatide.

A high cross-linking polystyrene(PSt)-based anion-exchange material with uniformly size, high ion exchange capacity, and high hydrophilicity was synthesized by a novel surface functionalization approach in this study. Uniformly sized PSt microspheres were prepared by the membrane emulsion polymerization strategy, and then modified by (1) conversing resid ual surface vinyl groups to epoxy groups followed by quaternization, and (2) decorating aromatic ring matrix including nitration, reduction and attachment of glycidyltrimethylammonium chloride. The 3-D morphology and porous features of microspheres were observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface of the modified PSt became roughness but the particle size remained same. Meanwhile, FT-IR spectra and laser scanning confocal microscope (LCSM) indicated that the modification groups had been successfully covalently coated onto the PSt microspheres. Modified PSt microspheres showed greatly improved hydrophilicity and biocompatibility with 0.387mmol/mL ion exchange capacity (IEC). In the application evaluation procedure, exenatide can be purified from 42.9% (peptide crudes) to 88.6% by modified PSt column with 97.1% recovery yield. This modified PSt microspheres had a large potential in application for efficient separation of peptides.

High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described.

Protease inhibitors from marine venomous animals and their counterparts in terrestrial venomous animals.

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared.

Connectivity maps for biosimilar drug discovery in venoms: the case of Gila monster venom and the anti-diabetes drug Byetta®.

Like most natural product libraries animal venoms have long been recognized as potentially rich source of biologically active molecules with the potential to be mined for the discovery of drugs, drug leads and/or biosimilars. In this work we demonstrate as a proof of concept a novel approach to explore venoms for potential biosimilarity to other drugs based on their ability to alter the transcriptomes of test cell lines followed by informatic searches and Connectivity Mapping to match the action of the venom on the cell gene expression to that of other drugs in the Connectivity Map (C-Map) database. As our test animal venom we chose Heloderma suspectum venom (Gila monster) since exendin-4, a glucagon-like peptide 1 receptor agonist, isolated from the venom is currently on the market to treat type 2 diabetes. The action of Byetta(®) (exentide, synthetic exendin-4), was also used in transcriptome studies. Analysis of transcriptomes from cells treated with the venom or the drug showed similarities as well as differences. The former case was primarily attributed to the fact that Gila monster venom likely contains a variety of biologically active molecules that could alter the MCF7 cell transcriptome compared to that of the single perturbant Byetta(®). Using Ingenuity Pathway Analysis software, insulin-like growth factor 1 signaling was identified in the category of "Top Canonical Pathways" for both the venom and Byetta(®). In the category of "Top Molecules" up-regulated, both venom and Byetta(®) shared IL-8, cyclic AMP-dependent transcription factor 3 (ATF-3), neuron-derived orphan receptor 1 (NR4A3), dexamethasone-induced Ras-related protein 1 (RASD1) and early growth response protein 1, (EGR-1) all with potential relevance in diabetes. Using Connectivity Mapping, Gila monster venom showed positive correlation with 1732 instances and negative correlation with 793 instances in the Connectivity database whereas Byetta(®) showed positive correlation with 1692 instances and negative correlation with 868 instances. Interestingly, the Gila monster venom and Byetta(®) both showed positive correlation with the anti-diabetic drugs troglitazone, of the thiazolidinedione class, and metformin, of the biguanide class, although Byetta(®) as a glucagon-like peptide-1 (GLP-1) agonist functions in a different manner than either of these two classes of anti-diabetic drugs. In summary, despite the fact that Gila monster venom contains a mixture of biologically active molecules, similarities in terms of perturbation of gene expression profiles on MCF7 cells were observed between the venom and the drug Byetta(®). Furthermore, using Connectivity Mapping the Gila monster venom was demonstrated to have nodes of positive correlation to several anti-diabetic drugs two of which were the same as observed with Byetta(®). Therefore, this study suggests that by using this approach novel drug activities heretofore unconsidered may be discovered in venoms using informatic tools and Connectivity Mapping.

Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus.

Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.

Antiplasmodial effect of the venom of Crotalus durissus cumanensis, crotoxin complex and Crotoxin B.

The antiplasmodial activity of phospholipases A(2) (PLA(2)) isolated from different animals has been studied. We explored the in vitro anti Plasmodium falciparum effect of a fraction containing crotoxin, Crotoxin B and whole venom of the rattlesnake Crotalus durissus cumanensis. Fraction II (crotoxin complex) was obtained by size exclusion chromatography, whereas Crotoxin B was purified by RP-HPLC. The whole venom is active against the parasite at concentrations of 0.17±0.03 µg/ml, fraction II at 0.76±0.17 µg/ml and Crotoxin B at 0.6±0.04 µg/ml. Differences were observed in the cytotoxic activity against peripheral mononuclear cells, with Crotoxin B exhibiting the highest cytotoxicity. The concentration of Crotoxin B required to exert cytotoxic activity was higher than that required to exert antiplasmodial activity. Lethality in mice confirmed the higher toxicity and neurotoxicity of whole venom and fraction II, whereas Crotoxin B was not lethal at the doses tested. These results suggest the potential of Crotoxin B as a lead compound for antimalarial activity.

Establishment and optimization of a wheat germ cell-free protein synthesis system and its application in venom kallikrein.

Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu(2+) were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/µl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu(2+) (5 mM) were added to the system and incubated at 26°C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies.

Towards an integrated venomics approach for accelerated conopeptide discovery.

Conopeptides and conotoxins are small peptides produced by cone snails as a part of their predatory/defense strategies that target key ion channels and receptors in the nervous system. Some of these peptides also potently target mammalian ion channels involved in pain pathways. As a result, these venoms are a source of valuable pharmacological and therapeutic agents. The traditional approach towards conopeptide discovery relied on activity-guided fractionation, which is time consuming and resource-intensive. In this review, we discuss the advances in the fields of transcriptomics, proteomics and bioinformatics that now allow researchers to integrate these three platforms towards a more efficient discovery strategy. In this review, we also highlight the challenges associated with the wealth of data generated with this integrated approach and briefly discuss the impact these methods could have on the field of toxinology.

Helokinestatin-7 peptides from the venoms of Heloderma lizards.

Helokinestatins 1-6 constitute a family of bradykinin antagonist peptides originally isolated from the venoms of the Gila Monster, Heloderma suspectum and the Mexican beaded lizard, Heloderma horridum. Here we report the identification, isolation and preliminary pharmacological characterization of two novel tridecapeptides, named helokinestatin-7S (FDDDSTELILEPR - 1550 Da) and helokinestatin-7H (FDDDSRKLILEPR - 1604 Da), whose primary structures were predicted from cDNAs cloned from venom libraries of respective Heloderma lizards. Computed molecular masses of putative helokinestatin-7 peptides were used as tools to locate these peptides in archived LC/MS fractions from respective venoms and sequences were confirmed by MS/MS fragmentation. A synthetic replicate of helokinestatin-7H was found to antagonize the relaxation effect of bradykinin on rat arterial smooth muscle but to have no measurable effects alone. In contrast, synthetic helokinestatin-7S was found to directly contract this preparation. Studies on related natural peptides with subtle differences in primary structure can provide the tools for structure/activity studies in pharmacological investigations directed toward unraveling the molecular basis of venom toxicity and for the evaluation of potential therapeutic leads.