Expression and purification of recombinant alpha-toxin AnCra1 from the scorpion Androctonus crassicauda and its functional characterization on mammalian sodium channels.

BACKGROUND: Alpha-scorpion toxins with long-chain peptide and four disulfide bonds represent diverse pharmacological profiles for various subtypes of voltage-gated sodium channels. Obtaining the natural toxins are difficult and time-consuming process, which represents the major difficulty to interpreting analysis of their structural and functional properties. METHODS AND RESULTS: This study describes the toxin peptide and plasmid construct containing the gene coding for mammalian toxin AnCra1 from the scorpion Androctonus crassicauda venom. We have established genetic construction of fusion protein in pET32a + vector containing thioredoxin (Trx-tag), enterokinase cleavage site and 6xhistidine-tag for efficient expression in Escherichia coli strain RG2 (DE3). The soluble expressed peptide, then purified by Ni-NTA resin affinity chromatography and its purity was confirmed by reverse-phase HPLC and mass spectrometry (7433.54 Da.). The electrophysiological data showed that recombinant AnCra1 selectively inhibits the fast inactivation of hNav1.7 channel (EC50 = 136.7 ± 6.6 nM). CONCLUSIONS: Our findings demonstrate that the AnCra1 is structurally and functionally analogous to alpha excitatory toxins; furthermore, expression and purification of bioactive scorpion toxins in bacterial cells can be a practicable and efficient way to obtain a novel source of toxin peptides as tools to study the function and physiological responses of ion channels.

Colombian Scorpion Centruroides margaritatus: Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel.

The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the humanether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC50 of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin É£-KTx1.1, isolated from Centruroides noxius, CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other É£-KTx.

Net charge tuning modulates the antiplasmodial and anticancer properties of peptides derived from scorpion venom.

VmCT1, a linear helical antimicrobial peptide isolated from the venom of the scorpion Vaejovis mexicanus, displays broad spectrum antimicrobial activity against bacteria, fungi, and protozoa. Analogs derived from this peptide containing single Arg-substitutions have been shown to increase antimicrobial and antiparasitic activities against Trypanossoma cruzi. Here, we tested these analogs against malaria, an infectious disease caused by Plasmodium protozoa, and assessed their antitumoral properties. Specifically, we tested VmCT1 synthetic variants [Arg]3 -VmCT1-NH2 , [Arg]7 -VmCT1-NH2 , and [Arg]11 -VmCT1-NH2 , against Plasmodium gallinaceum sporozoites and MCF-7 mammary cancer cells. Our screen identified peptides [Arg]3 -VmCT1-NH2 and [Arg]7 -VmCT1-NH2 as potent antiplasmodial agents (IC50 of 0.57 and 0.51 µmol L-1 , respectively), whereas [Arg]11 -VmCT1-NH2 did not show activity against P. gallinaceum sporozoites. Interestingly, all peptides presented activity against MCF-7 and displayed lower cytotoxicity toward healthy cells. We demonstrate that increasing the net positive charge of VmCT1, through arginine substitutions, modulates the biological properties of this peptide family yielding novel antiplasmodial and antitumoral molecules.

Novel scorpion venom peptide HsTx2 ameliorates cerebral ischemic brain injury in rats via the MAPK signaling pathway.

Ischemic stroke is a severe threat to human health due to its high recurrence, mortality, and disability rates. As such, how to prevent and treat ischemic stroke effectively has become a research hotspot in recent years. Here, we identified a novel peptide, named HsTx2 (AGKKERAGSRRTKIVMLKCIREHGH, 2 861.855 Da), derived from the scorpion Heterometrus spinifer, which showed obvious anti-apoplectic effects in rats with ischemic stroke. Results further demonstrated that HsTx2 significantly reduced formation of infarct area and improved behavioral abnormalities in ischemic stroke rats. These protective effects were likely exerted via activation of the mitogen-activated protein kinase (MAPK) signaling pathway, i.e., up-regulation of phosphorylated ERK1/2 in both rat cerebral cortex and activated microglia (AM); up-regulation of phosphorylated p38 (p-p38) in the cerebral cortex; and inhibition of phosphorylated JNK and p-p38 levels in the AM. In conclusion, this study highlights HsTx2 as a potential neuroprotective agent for stroke.

Pioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase.

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Nav1.4, Nav1.6, and BgNav1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Bradykinin-potentiating factor isolated from Leiurus quinquestriatus scorpion venom alleviates cardiomyopathy in irradiated rats via remodelling of the RAAS pathway.

The objective of this work was to evaluate the effect of a bradykinin-potentiating factor (BPF) isolated from Leiurus quinquestriatus scorpion venom as a natural modulator of radiation-induced cardiac damage. Four groups of rats were treated as follows; control group, group receiving BPF (1 µg/g b.wt i.p./biweekly) for 4 weeks, group irradiated at 6 Gy, group receiving BPF post-irradiation for 4 weeks. Irradiation induced a significant elevation of myocardial parameters: atrial natriuretic peptide (ANP), cardiac troponin I (cTnI), potassium (K+ ) and creatine kinase (CK); vascular indices: lactate dehydrogenase (LDH), inducible nitric oxide synthase (iNOS) and endothelin I; oxidative stress indices: malondialdehyde (MDA) associated with a significant depletion of both reduced glutathione (GSH) in the cardiac tissue homogenate and serum ferric reducing antioxidant power (FRAP) depletion and significantly reinforced elevation of Renin Angiotensin Aldosterone System (RAAS) indices: serum angiotensin II (AngII) and aldosterone, and also protein expression of cleaved caspase-3 and cyclophilin A. BPF administration altered the biochemical damage of radiation, specifically inhibited AngII formation, aldosterone release and prevented the histopathological and immunohistochemical alterations which were observed in cardiac tissue with significant reduction in mean arterial blood pressure (MAP) caused by irradiation. In conclusion, biochemical assays, histopathological and immunohistochemical findings of the present study demonstrated that exogenous BPF isolated from scorpion venom reduced the cardiomyopathy alterations induced by irradiation via remodelling of the RAAS pathway.

IsCT-based analogs intending better biological activity.

IsCT1-NH2 is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α-helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)-based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out. Peptides with a substitution at position 3 or 5 ([L]3 -IsCT1-NH2 , [K]3 -IsCT1-NH2 , or [F]5 -IsCT1-NH2 ) showed no significant change in an activity relative to IsCT1-NH2 . The addition of a proline residue at position 8 ([P]8 -IsCT1-NH2 ) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13-50 µmol L-1 ), and the addition of a tryptophan residue at position 1 ([W]1 -IsCT1-NH2 ) increased the hemolytic activity (MHC = 1.56 µmol L-1 ) without an improvement in antimicrobial activity. The analog [A]1 [F]5 [K]8 -IsCT1-NH2 , which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 µmol L-1 ) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.

Structural and functional characterization of toxic peptides purified from the venom of the Colombian scorpion Tityus macrochirus.

The soluble venom of the scorpion Tityus macrochirus was separated by chromatographic procedures and three homogeneous peptides were obtained and their primary structures were determined. They were called: Tma1-Tma3, from the abbreviated name of the scorpion. Tma1 is a peptide containing 65 amino acids with four disulfide linkages and a molecular weight of 7386.2 Da. It is a mammalian toxin, shown to affect human sodium-channels sub-types hNav1.6 and hNav1.4. Tma2 and Tma3 are peptides containing 69 amino acids linked by four disulfide bonds, molecular weights 7819.7 and 7830.0 Da, respectively. They do not affect human sodium-channels but are lethal to insects (crickets). A phylogenic analysis of the three peptides and those of other toxic peptides isolated from the genus Tityus and Centruroides were grouped together and analyzed, permitting to obtain a topology with two main clades, one includes most sodium-channel anti-insect scorpion toxins and others includes mostly sodium-channel scorpion toxins anti-mammalian. Tma1 segregates among a group of well-studied ß-class toxins of other Tityus species such as T. discrepans, T. obscurus and T. pachyurus. Tma2 and Tma3 are associated with anti-insect toxins, particularly with one of T. obscurus. This phylogenetic analysis confirms and enforces our experimental results obtained with these three new sodium-channel scorpion toxins.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Recombinant Production and Structure-Function Study of the Ts1 Toxin from the Brazilian Scorpion Tityus serrulatus.

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.

Chlorotoxin targets ERα/VASP signaling pathway to combat breast cancer.

Breast cancer is one of the most common malignant tumors among women worldwide. About 70-75% of primary breast cancers belong to estrogen receptor (ER)-positive breast cancer. In the development of ER-positive breast cancer, abnormal activation of the ERα pathway plays an important role and is also a key point leading to the failure of clinical endocrine therapy. In this study, we found that the small molecule peptide chlorotoxin (CTX) can significantly inhibit the proliferation, migration and invasion of breast cancer cells. In in vitro study, CTX inhibits the expression of ERα in breast cancer cells. Further studies showed that CTX can directly bind to ERα and change the protein secondary structure of its LBD domain, thereby inhibiting the ERα signaling pathway. In addition, we also found that vasodilator stimulated phosphoprotein (VASP) is a target gene of ERα signaling pathway, and CTX can inhibit breast cancer cell proliferation, migration, and invasion through ERα/VASP signaling pathway. In in vivo study, CTX significantly inhibits growth of ER overexpressing breast tumor and, more importantly, based on the mechanism of CTX interacting with ERα, we found that CTX can target ER overexpressing breast tumors in vivo. Our study reveals a new mechanism of CTX anti-ER-positive breast cancer, which also provides an important reference for the study of CTX anti-ER-related tumors.

Study on the obtaining of Tityus trivittatus venom in Argentina.

Envenomation by scorpions of the genus Tityus is an important public health problem in Argentina, involving near 8000 stings and 2 deaths each year. Treatment for envenomation is the use of specific antivenom and intensive hospital care. Antivenom is produced by the Ministry of Health and freely distributed throughout the country. For antivenom production it is necessary to collect scorpion venom, which is a difficult task because although scorpions can be found in Argentina, they are less abundant than in warmer latitudes. For this reason venom collection constitutes a bottleneck for antivenom production. Although in Argentina several species of Tityus can be found, most of the accidents are caused by Tityus trivittatus, and the venom of this scorpion has historically been the venom used for antivenom production. We analyzed retrospectively 26 pools of telson homogenates (6964 telsons) and 37 pools of milked venom obtained by electrical stimulation (equivalent to 6841 milkings). Lethal potencies of samples from different provinces were very similar, although venom from scorpions of Buenos Aires city showed the lowest potency. The venom obtained by milking (median LD50 12.3 µg), provided batches containing LD50s more potent when compared with the venom obtained from telson homogenates (p < 0.0001). Many batches of telson homogenates (30%) showed lower potencies than acceptable for antivenom production and control. In addition to the study of the venom yield, the records of immunization of horses, the potency of the batches and the protein content of each batch of anti-scorpion antivenom produced were analyzed, comparing those produced using milked venom with those using telson homogenates as immunogens. Batches produced using milked venom required a shorter period of immunization (p < 0.0001), rendered higher neutralizing titers (p 0.0350) and possessed lower protein content (p 0.0092). Results clearly showed that the milking of scorpions is a more efficient tool to obtain venom for antivenom production in comparison to the use of telson homogenates.

Recombinant scorpion toxins: Focus on four-disulfide peptide blockers of Kv1-channels.

We have recently developed a simple and effective bioengineering approach to large-scale production of alpha-KTx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. This approach was successfully approved for different peptides containing three disulfide bonds. To extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction with tobacco etch virus (TEV) protease need to be found. For this, the interplay between efficiency of TEV hydrolysis and sensitivity of the target peptides to disulfide reducing agents was studied, and optimized protocols of TEV cleavage reaction were worked out. Maurotoxin and hetlaxin were produced in a folded form avoiding in vitro renaturation step with yields of 14 and 12 mg/liter of culture, respectively.

Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels.

Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain α-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7±6.6nM for rNav1.4, 454.2±50.1nM for hNav1.5 and 30.9±10.3µM for hNav1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNav1.4 and hNav1.5 channels toward a more negative direction and present the typical features of a ß-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNav1.4 and hNav1.5 channels. The V1/2 values of the steady-state inactivation were altered to be more positive for rNav1.4 and more negative for hNav1.5. Moreover, the recovery of the hNav1.5 channel from inactivation was more significantly delayed than that of the rNav1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both α- and ß-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.

Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli.

Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli. Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl ß-d-1-thiogalactopyranoside, and it was purified by Ni2+-nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust (Locusta migratoria) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests.

Mass fingerprinting and electrophysiological analysis of the venom from the scorpion Centruroides hirsutipalpus (Scorpiones: Buthidae)

Centruroides hirsutipalpus, of the family Buthidae, is a scorpion endemic to the Western Pacific region of Mexico. Although medically important, its venom has not yet been studied. Therefore, this communication aims to identify their venom components and possible functions. Methods Fingerprinting mass analysis of the soluble venom from this scorpion was achieved by high-performance liquid chromatography and electrospray mass spectrometry. Furthermore, the soluble venom and its toxic effects were evaluated extensively via electrophysiological assays in HEK cells expressing human voltage-gated Na+ channels (hNav 1.1 to Nav1.6), CHO cells expressing hNav 1.7, potassium channel hERG 1 (Ether-à-go-go-related-gene) and the human K+-channel hKv1.1. Results The separation of soluble venom produced 60 fractions from which 83 distinct components were identified. The molecular mass distribution of these components varies from 340 to 21,120 Da. Most of the peptides have a molecular weight between 7001 and 8000 Da (46% components), a range that usually corresponds to peptides known to affect Na+ channels. Peptides with molecular masses from 3000 to 5000 Da (28% of the components) were identified within the range corresponding to K+-channel blocking toxins. Two peptides were obtained in pure format and completely sequenced: one with 29 amino acids, showing sequence similarity to an "orphan peptide" of C. limpidus, and the other with 65 amino acid residues shown to be an arthropod toxin (lethal to crustaceans and toxic to crickets). The electrophysiological results of the whole soluble venom show a beta type modification of the currents of channels Nav1.1, Nav1.2 and Nav1.6. The main effect observed in channels hERG and hKv 1.1 was a reduction of the currents. Conclusion The venom contains more than 83 distinct components, among which are peptides that affect the function of human Na+-channels and K+-channels. Two new complete amino acid sequences were determined: one an arthropod toxin, the other a peptide of unknown function.(AU)

Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus.

The pallid bat (Antrozous pallidus), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, Centruroides sculpturatus. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD50 concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance.

Evaluation of the in vitro antimicrobial activity of selected Saudi scorpion venoms tested against multidrug-resistant micro-organisms.

OBJECTIVES: Scorpion venoms are a rich source of bioactive peptides with promising clinical value that may lead to the discovery and development of new drugs. The present study was designed to evaluate the in vitro antimicrobial activities of the venoms extracted from three medically important Saudi scorpions (Androctonus crassicauda, Androctonus bicolor and Leiurus quinquestriatus). METHODS: Antimicrobial assays were performed using a microplate growth inhibition assay against 10 multidrug-resistant (MDR) micro-organisms (4 Gram-negative bacteria, 2 Gram-positive bacteria and 4 fungi and yeasts) at concentrations ranging from 0 to 20mg/mL of each venom. Following qualitative analysis, dose-response assays were performed for bacterial and fungal killing curves using the MTT colorimetric assay. RESULTS: Among the three tested scorpion venoms, only L. quinquestriatus venom showed significant broad-spectrum antimicrobial activity in a dose-dependent manner from 5 to 20mg/mL. Leiurus quinquestriatus venom inhibited the growth and survival of MDR Escherichia coli (55.2%), Acinetobacter baumannii (50.6%), Klebsiella pneumoniae (35.1%), Pseudomonas aeruginosa (31.3%), Staphylococcus aureus (36.4%), Enterococcus faecalis (47.6%), Candida albicans (31.2%) and Candida glabrata (39.0%), whereas no significant activity against Fusarium oxysporum and Aspergillus flavus was observed. In contrast, the venoms of A. crassicauda and A. bicolor did not show noticeable antimicrobial activity against any of the tested organisms. CONCLUSIONS: The findings of the current study demonstrate that L. quinquestriatus venom possesses antimicrobial activity and thus can be used as a template for designing and development of novel antimicrobial drugs.

Purificación parcial de péptidos presentes en el veneno del escorpión Tityus macrochirus (Buthidae) y evaluación preliminar de su actividad citotóxica / Partial purification of peptides present in the Tityus macrochirus (Buthidae) scorpion venom and preliminary assessment of their cytotoxicity

RESUMEN Introducción. El veneno del escorpión posee péptidos con actividad neurotóxica que actúan principalmente en los canales iónicos del sistema nervioso de insectos y mamíferos. También se ha establecido su acción citolítica y anticancerígena, características biológicas que aún no se han explorado en el veneno del escorpión Tityus macrochirus. Objetivo. Evaluar si tanto el veneno total de T. macrochirus como la fracción de péptidos parcialmente purificados disminuyen el porcentaje de viabilidad de diferentes líneas celulares provenientes de tumores. Materiales y métodos. Mediante métodos cromatográficos, electroforéticos y de ultrafiltración con membranas de Amicon Ultra 0.5®, se identificaron y purificaron parcialmente los péptidos del veneno de T. macrochirus obtenido mediante estimulación eléctrica. Los ensayos de actividad citotóxica del veneno y de la fracción de péptidos se hicieron en líneas celulares provenientes de tumores con el método colorimétrico de reducción de la sal de tetrazolio (Mossman's Tetrazole Test, MTT). Resultados. El veneno de T. macrochirus posee péptidos con pesos moleculares entre 3 y 10 kDa, los cuales se purificaron parcialmente mediante ultrafiltración y se evaluaron mediante cromatografía líquida de alta resolución en fase inversa (Reverse Phase-High Pressure Liquid Chromatography, RP-HPLC). Los ensayos de citotoxicidad del veneno total de T. macrochirus evidenciaron una mayor disminución de la viabilidad en la línea celular PC3 que en las demás líneas celulares evaluadas, en tanto que la fracción parcialmente purificada de péptidos logró disminuir la viabilidad de la línea celular HeLa. Conclusión. Los péptidos del veneno de T. macrochirus presentaron actividad citotóxica en algunas de las líneas celulares provenientes de tumores, y se observó algún grado de selectividad frente a ellas.

ABSTRACT Introduction: Scorpion venom contains peptides with neurotoxic action primarily active on ion channels in the nervous system of insects and mammals. They are also characterized as cytolytic and anticancer, biological characteristics that have not yet been reported for the Tityus macrochirus venom. Objective: To assess if the total T. macrochirus venom and the fraction of partially purified peptides decrease the viability of various tumor-derived cell lines. Materials and methods: The scorpion venom was collected by electrical stimulation and, subsequently, subjected to chromatography, electrophoresis, and ultrafiltration with Amicon Ultra 0.5® membranes for the partial identification and purification of its peptides. The cytotoxic activity of the venom and the peptides fraction trials on tumor-derived cell lines were carried out by the MTT method. Results: The T. macrochirus scorpion venom has peptides with molecular weights ranging between 3 and 10 kDa. They were partially purified using the ultrafiltration technique, and assessed by the RP-HPLC method. Cytotoxicity trials with the whole T. macrochirus venom showed a higher viability decrease on the PC3 cell line compared to the other cell lines assessed, while the partially purified peptides decreased the HeLa cell line viability. Conclusion: Peptides in the T. macrochirus scorpion venom showed cytotoxic activity on some tumor-derived cell lines. We observed some degree of selectivity against other cell lines assessed.