BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization.

Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.

Biodegradable microparticles containing crotamine isolated from Crotalus durissus terrificus display antileishmanial activity in vitro.

BACKGROUND/AIMS: To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro. METHODS: Particles were analyzed on-chip by surface plasmon resonance and characterized by testing their diameters, zeta potential and encapsulation rate. The viability of promastigotes as well as murine macrophages was assessed. Furthermore, the phagocytic index was determined for macrophages, and cell supernatants were collected for the determination of TNF-α levels. An infection assay using Leishmania amazonensis-infected macrophages was also conducted. RESULTS: The diameters and zeta potential of control particles (1.35 µm; -12.3 mV) and of those containing crotamine (3.09 µm; -20.9 mV) were adequate for the assays conducted. Crotamine-loaded particles were better captured by macrophages than control particles (increase of 12% in the phagocytic index), leading to increased TNF-α levels (196 pg/ml), and they also induced a significant decrease in the numbers of amastigotes compared to infected macrophages only. CONCLUSION: The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará.

BACKGROUND: The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. METHODS: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. RESULTS: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. CONCLUSION: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.

Antimalarial activity of basic phospholipases A2 isolated from Paraguayan Bothrops diporus venom against Plasmodium falciparum.

Malaria is a parasitic infectious disease and was responsible for 400.000 deaths in 2018. Plasmodium falciparum represents the species that causes most human deaths due to severe malaria. In addition, studies prove the resistance of P. falciparum to drugs used to treat malaria, making the search for new drugs with antiplasmodial potential necessary. In this context, the literature describes snake venoms as a rich source of molecules with microbicidal potential, including phospholipases A2 (PLA2s). In this sense, the present study aimed to isolate basic PLA2s from Paraguayan Bothrops diporus venom and evaluate their antiplasmodial potential. Basic PLA2s were obtained using two chromatographic steps. Initially, B. diporus venom was subjected to ion exchange chromatography (IEC). The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the PLA2s. The toxins were tested for enzymatic activity using a chromogenic substrate and finally, the antiplasmodial, cytotoxic potential and hemolytic activity of the isolated toxins were evaluated. The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the two enzymatically active PLA2s, BdTX-I and BdTX-II and the PLA2 homologue BdTX-III. The antiplasmodial potential was evaluated and the toxins showed IC50 values of: 2.44, 0.0153 and 0.59 µg/mL respectively, presenting PLA2 selectivity according to the selectivity index results (SI) calculated against HepG2 cells. The results show that the 3 basic phospholipases isolated in this study have a potent antiparasitic effect against the W2 strain of P. falciparum. In view of the results obtained in this work, further research are necessary to determine the mechanism of action by which these toxins cause cell death in parasites.

Isolation, Biochemical Characterization and Antiparasitic Activity of BmatTX-IV, A Basic Lys49-Phospholipase A2 from the Venom of Bothrops mattogrossensis from Paraguay.

BACKGROUND: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. METHODS: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. RESULTS: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 µg/mL against Leishmania infantum promastigotes and of 13.8 µg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 µg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 µg/mL. CONCLUSION: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.

Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae).

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Corrigendum to "Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae)".

[This corrects the article DOI: 10.1155/2019/2492315.].

Biochemical characterization of a phospholipase A2 homologue from the venom of the social wasp Polybia occidentalis.

BACKGROUND: Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. METHODS: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. RESULTS: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896.47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. CONCLUSION: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.

Pharmacological characterization of cnidarian extracts from the Caribbean Sea: evaluation of anti-snake venom and antitumor properties.

BACKGROUND: Cnidarians produce toxins, which are composed of different polypeptides that induce pharmacological effects of biotechnological interest, such as antitumor, antiophidic and anti-clotting activities. This study aimed to evaluate toxicological activities and potential as antitumor and antiophidic agents contained in total extracts from five cnidarians: Millepora alcicornis, Stichodactyla helianthus, Plexaura homomalla, Bartholomea annulata and Condylactis gigantea (total and body wall). METHODS: The cnidarian extracts were evaluated by electrophoresis and for their phospholipase, proteolytic, hemorrhagic, coagulant, fibrinogenolytic, neuromuscular blocking, muscle-damaging, edema-inducing and cytotoxic activities. RESULTS: All cnidarian extracts showed indirect hemolytic activity, but only S. helianthus induced direct hemolysis and neurotoxic effect. However, the hydrolysis of NBD-PC, a PLA2 substrate, was presented only by the C. gigantea (body wall) and S. helianthus. The extracts from P. homomalla and S. helianthus induced edema, while only C. gigantea and S. helianthus showed intensified myotoxic activity. The proteolytic activity upon casein and fibrinogen was presented mainly by B. annulata extract and all were unable to induce hemorrhage or fibrinogen coagulation. Cnidarian extracts were able to neutralize clotting induced by Bothrops jararacussu snake venom, except M. alcicornis. All cnidarian extracts were able to inhibit hemorrhagic activity induced by Bothrops moojeni venom. Only the C. gigantea (body wall) inhibited thrombin-induced coagulation. All cnidarian extracts showed antitumor effect against Jurkat cells, of which C. gigantea (body wall) and S. helianthus were the most active; however, only C. gigantea (body wall) and M. alcicornis were active against B16F10 cells. CONCLUSION: The cnidarian extracts analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms; these results may contribute to elucidate the possible mechanisms of interaction between cnidarian extracts and snake venoms.

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX, a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766).

Snake venom phospholipases A2 (PLA2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 µg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.

Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes.

Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIß and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment.

BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential.

Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100µg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64µg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150µg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom.

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI-5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms.

Antitumoral Potential of Snake Venom Phospholipases A2 and Synthetic Peptides.

Cancer, a disease that currently affects approximately 14 million people, is characterized by abnormal cell growth with altered replication capacity, which leads to the development of tumor masses without apoptotic control. Resistance to the drugs used in chemotherapy and their side effects stimulate scientific research seeking new therapies to combat this disease. Molecules from flora and fauna with cytotoxic activity against tumor cells have been studied for their potential to become a source of pharmaceutical agents. In this regard, snake venoms have a variety of proteins and peptides that have proven biotechnological potential. In several studies, antibacterial action and antitumor activity have been observed. One of the most widely studied venom components are phospholipases A2. Snake venom phospholipases A2 (svPLA2s) comprise a large class of molecules that catalyze the hydrolysis of the sn-2 position of phospholipids releasing fatty acids and lysophospholipids and are related to a broad spectrum of biotechnological activities. In addition to their specific cytotoxicity against some tumor cell lines, inhibitory activity of angiogenesis, adhesion and cell migration has been described. The antitumor activity of svPLA2s was observed both in vitro and in vivo, but little is known about the mechanism of action of these proteins in promoting this activity. In this review, the main structural and functional characteristics of svPLA2s are discussed, along with the mechanisms proposed, thus far, to explain their antitumor activity, targeting their potential use as a therapeutic alternative against cancer.

Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.

Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 µg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications.

Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

BbMP-1, a new metalloproteinase isolated from Bothrops brazili snake venom with in vitro antiplasmodial properties.

This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bß chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC50 of 3.2 ± 2.0 µg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity.

Biological characterization of the Amazon coral Micrurus spixii snake venom: Isolation of a new neurotoxic phospholipase A2.

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 µg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential.