Synthetic Peptide Derived from Scorpion Venom Displays Minimal Toxicity and Anti-infective Activity in an Animal Model.

Multidrug-resistant bacteria represent a global health problem increasingly leading to infections that are untreatable with our existing antibiotic arsenal. Therefore, it is critical to identify novel effective antimicrobials. Venoms represent an underexplored source of potential antibiotic molecules. Here, we engineered a peptide (IsCT1-NH2) derived from the venom of the scorpion Opisthacanthus madagascariensis, whose application as an antimicrobial had been traditionally hindered by its high toxicity. Through peptide design and the knowledge obtained in preliminary studies with single and double-substituted analogs, we engineered IsCT1 derivatives with multiple amino acid substitutions to assess the impact of net charge on antimicrobial activity and toxicity. We demonstrate that increased net charge (from +3 to +6) significantly reduced toxicity toward human erythrocytes. Our lead synthetic peptide, [A]1[K]3[F]5[K]8-IsCT1-NH2 (net charge of +4), exhibited increased antimicrobial activity against Gram-negative and Gram-positive bacteria in vitro and enhanced anti-infective activity in a mouse model. Mechanism of action studies revealed that the increased antimicrobial activity of our lead molecule was due, at least in part, to its enhanced ability to permeabilize the outer membrane and depolarize the cytoplasmic membrane. In summary, we describe a simple method based on net charge tuning to turn highly toxic venom-derived peptides into viable therapeutics.

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.

Mygalin: An Acylpolyamine With Bactericidal Activity.

Inappropriate use of antibiotics favors the selection and spread of resistant bacteria. To reduce the spread of these bacteria, finding new molecules with activity is urgent and necessary. Several polyamine analogs have been constructed and used to control microorganisms and tumor cells. Mygalin is a synthetic acylpolyamine, which are analogs of spermidine, derived from the hemolymph of the spider Acanthoscurria gomesiana. The effective activity of polyamines and their analogs has been associated with their structure. The presence of two acyl groups in the Mygalin structure may give this molecule a specific antibacterial activity. The aim of this study was to identify the mechanisms involved in the interaction of Mygalin with Escherichia coli to clarify its antimicrobial action. The results indicated that Mygalin exhibits intense dose and time-dependent bactericidal activity. Treatment of E. coli with this molecule caused membrane rupture, inhibition of DNA synthesis, DNA damage, and morphological changes. The esterase activity increased along with the intracellular production of reactive oxygen species (ROS) after treatment of the bacteria with Mygalin. In addition, this molecule was able to sequester iron and bind to LPS. We have shown that Mygalin has bactericidal activity with underlying mechanisms involving ROS generation and chelation of iron ions that are necessary for bacterial metabolism, which may contribute to its microbicidal activity. Taken together, our data suggest that Mygalin can be explored as a new alternative drug with antimicrobial potential against Gram-negative bacteria or other infectious agents.

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells.

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

Human Antimicrobial Peptide Isolated From Triatoma infestans Haemolymph, Trypanosoma cruzi-Transmitting Vector.

The importance of antimicrobial peptides (AMPs) in relation to the survival of invertebrates is well known. The source and the mode of action on the insects' immune system of these molecules have been described from different perspectives. Insects produce their own AMPs as well as obtain these molecules from various sources, for example by absorption through the intestinal tract, as previously described for Boophilus microplus. Blood-sucking barber bug Triatoma infestans attracts social, economic and medical interest owing to its role in the transmission of Chagas disease. Despite new studies, descriptions of AMPs from this insect have remained elusive. Thus, the aims of this work were to characterize the antimicrobial potential of human fibrinopeptide A (FbPA) obtained from the T. infestans haemolymph and identify its natural source. Therefore, FbPA was isolated from the T. infestans haemolymph through liquid chromatography and identified by mass spectrometry. This peptide exhibited antimicrobial activity against Micrococcus luteus. Native FbPA from human blood and the synthetic FbPA also exhibited antimicrobial activity. The synthetic FbPA was conjugated with fluorescein isothiocyanate and offered to the insects. The haemolymph collected after 72 h exhibited fluorescence at the same wavelength as fluorescein isothiocyanate. Our experiments show that beyond intrinsic AMP production, T. infestans is able to co-opt molecules via internalization and may use them as AMPs for protection.

Analysis of antioxidant, cytotoxic and mutagenic potential of Agave sisalana Perrine extracts using Vero cells, human lymphocytes and mice polychromatic erythrocytes.

Brazilian Northeast is the world's largest producer of Agave sisalana Perrine for the supply of the sisal fiber. About 95% of plant biomass, which comprise the mucilage and sisal juice, is considered a waste residual is discarded in the soil. However, the sisal juice is rich in steroidal saponins, which exhibits different pharmacological properties. Despite this, natural products are not necessarily safe. Based on this, this study analyzed the antioxidant, cytotoxic and mutagenic potential of three extracts derived from acid hydrolysis (AHAS), dried precipitate (DPAS) and hexanic of A. sisalana (HAS). These analyses were performed by in vitro and in vivo methods, using Vero cells, human lymphocytes and mice. Results showed that AHAS 50 and 100 can be considered a useful antineoplastic candidate due to their antioxidant and cytotoxic activity, with no genotoxic/clastogenic potential in Vero cells and mice. Although the comet assay in human lymphocytes has showed that the AHAS 25, AHAS 50 and AHAS 100 can lead to DNA breaks, these extracts did not promote DNA damages in mice bone marrow. Considering the different mutagenic responses obtained with the different methods employed, this study suggest that the metabolizing pathways can produce by-products harmful to health. For this reason, it is mandatory to analyze the mutagenic potential by both in vitro and in vivo techniques, using cells derived from different species and origins.

Antimicrobial activity of leucine-substituted decoralin analogs with lower hemolytic activity.

Linear cationic α-helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine-substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α-helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]10 -Dec-NH2 analog showed similar activity against different microorganisms (c.a. 0.4-0.8 µmol L-1 ), helical structuration, and some hemolytic activity, [Leu]8 -Dec-NH2 analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 µmol L-1 ). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Novel designed VmCT1 analogs with increased antimicrobial activity.

Antimicrobial peptides are biologically active molecules produced by a wide range of organisms as an essential component of the innate immune response. They have recently attracted great interest, since they have antimicrobial activity against a broad spectrum of microorganisms. VmCT1 is a cationic peptide from the venom of Vaejovis mexicanus smithi scorpions, which presents antibacterial activity and tends to helical structures. Its analogs were synthesized, characterized and the conformational studies were performed by circular dichroism. The peptides were designed to verify if the single and double substitutions proposed at the hydrophilic and hydrophobic portions of the amphipathic structure would alter antimicrobial activity against Gram-negative and Gram-positive bacteria, yeast and filamentous fungus, besides the hemolytic activity in human erythrocytes. Total charge of the peptides were modified from +2 to +3 by the introduction of a Lysine residue in the hydrophilic face of the amphiphilic helical structure leading to enhanced antimicrobial activity. [K]11-VmCT1-NH2 presented the lower MIC value against the microorganisms (from 0.39 to 6.25 µmol L-1), however it showed higher hemolytic activity. The other Lysine-substituted analogs presented also lower MIC values ranging from 0.39 to 25 µmol L-1 for the microorganisms assessed. The circular dichroism spectra analyses suggest that the Lysine-substituted analogs tend to adopt helical structures in trifluoroethanol solution and vesicles (fH: 0.43-1), however they were coiled in water. Alanine substitution by a Glutamic acid residue in the hydrophilic face promotes the increase of polar angle in [E]4-VmCT1-NH2 analog, which was important to led lower hemolytic activity (MHC value = 25 µmol L-1). [W]9-VmCT1-NH2 and [E]4[W]9-VmCT1-NH2 were designed to favors hydrophobic interactions by the introduction of Tryptophan residue. [W]9-VmCT1-NH2 presented MIC values lower or similar than the model molecule in the most of microorganisms tested. These results provided information about the structure-activity relationship and showed the influence of physicochemical parameters on antimicrobial and hemolytic activity.

VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae).

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC50; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.

Potamotrygon cf. henlei stingray mucus: biochemical features of a novel antimicrobial protein.

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons. In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the ß-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

The spider acylpolyamine Mygalin is a potent modulator of innate immune responses.

Mygalin is an antibacterial molecule isolated from the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-γ synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-γ activation. In addition, Mygalin-activated macrophages produced TNF-α but not IL-1ß, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-γ and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection.

Specialization of the sting venom and skin mucus of Cathorops spixii reveals functional diversification of the toxins.

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.

Studies on pharmacological properties of mucus and sting venom of Potamotrygon cf. henlei.

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout the rivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posing a serious public health problem even if not properly reported. The accidents usually involve fishermen and bathers, and to date there is no effective treatment for the injured. Considering these facts and limitations of studies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describe the principal pharmacological and certain biochemical properties of the mucus and sting venom. We found that mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Our results also indicate that the inflammatory cellular influx observed could be triggered by the venom and mucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested for antimicrobial activity in which only the mucus presented activity. It could be inferred from the present study that P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically active components.