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Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: KM 17 μM, kcat 1.2 s−1, VMax 2.2 U/mg and kcat/KM 7 × 10−4; for MESG they were: KM 131 μM, kcat 2.4 s−1, VMax 4.4 U/mg and kcat/KM 1.8 × 10−4. The thermodynamic parameters for the substrate Phosphate were: ΔG − 5.8 cal mol−1, ΔH − 6.5 cal mol−1 and ΔS − 2.25 cal mol−1/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.

Made available in DSpace on 2022-04-28T19:41:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-31 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10−7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound. Instituto de Genética e Bioquímica Universidade Federal de Uberlândia UFU Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista UNESP Laboratório de Biotecnologia de Proteínas e Compostos Bioativos LABIOPROT Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Laboratório de Imunologia Celular Aplicada à Saúde Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis IBAMA Departamento de Química Universidade Federal de Lavras UFLA Instituto Federal Goiano IF Goiano Faculty of Chemical Sciences University of Cuenca LAQV/Requimte Faculty of Sciences University of Porto Centro Universitário São Lucas UniSL Instituto Nacional de Ciência e Tecnologia em Epidemiologia da Amazônia Ocidental (INCT-EpiAmO) Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista UNESP

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a K

Made available in DSpace on 2020-12-12T01:29:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Secretaría Nacional de Ciencia, Tecnología e Innovación A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins. Faculdade de Ciências Farmacêuticas de Ribeirão Preto FCFRP Universidade de São Paulo USP Laboratório de Biotecnologia de Proteínas e Compostos Bioativos da Amazônia Ocidental LaBioProt Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia e Universidade Federal de Rondônia UNIR Centro de Informaciones e Investigaciones Toxicológicas y Químicas Aplicadas (CEIITOXQUIA) and Departamento de Química FCNYE Universidad Autónoma de Chiriquí UNACHI Instituto Brasileiro Do Meio Ambiente e Dos Recursos Naturais Renováveis IBAMA Unidade de Biotecnologia Universidade de Ribeirão Preto UNAERP Instituto Federal de Goiás IFG Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Laboratório de Imunologia Celular Aplicada a Saúde Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia Centro Universitário São Lucas UniSL Instituto Nacional de Ciência e Tecnologia Em Epidemiologia da Amazônia Ocidental INCT – EpiAmO Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP

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 ofR. marinasecretion 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 againstStaphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli,Plasmodium falciparum,Leishmania guyanensis, andLeishmania 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 ofR.marinasecretion as well as the bioactive potential of these molecules.

Made available in DSpace on 2018-12-11T16:47:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 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 100 μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14 ± 0.08 and 6.41 ± 0.64 μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64 ± 7.94 and >150 μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Fiocruz Rondônia Departamento de Medicina Universidade Federal de Rondônia UNIR Centro para el Desarrollo de la Investigación Científica CEDIC Laboratório da Plataforma de Bioensaios de Malária e Leishmaniose Fiocruz Rondônia Faculdade São Lucas FSL Departamento de Física e Biofísica Universidade Estadual Paulista UNESP Laboratório de Bioquímica e Biofísica Instituto Butantan Departamento de Física e Biofísica Universidade Estadual Paulista UNESP

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.

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

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.

Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB throughWestern blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.

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.

Background Research involving snake venom has gradually surpassed the simple discovery of new molecules using purification and structural characterization processes, and extended to the identification of their molecular targets and the evaluation of their therapeutic potential. Nevertheless, this only became possible due to constant progress in experimental biology and protein purification approaches. Objective This review aims to discuss the main components of snake venoms that have been investigated for biotechnological purposes, and to discover how these promising biomolecules were obtained with the satisfactory degree of purity that have enabled such studies. Advances in purification technologies of various snake venom molecules have allowed for important discoveries of proteins and peptides with different biomedical and biotechnological applications. Result and conclusion It is believed that significant experimental and computational advances will arise in similar proportions in the coming years that will allow researchers to map the molecular regions responsible for their pharmacological actions, their respective mechanisms of action and their cell targets.

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.

Crude venom ofBothrops jararacussuand isolated phospholipases A2(PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) byp-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2(PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Snake venoms contain various proteins, especially phospholipases A

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.

Background 3beta,6beta,16beta-trihydroxylup-20(29)-ene is a lupane triterpene isolated from Combretum leprosum fruit. The lupane group has been extensively used in studies on anticancer effects; however, its possible activity against protozoa parasites is yet poorly known. The high toxicity of the compounds currently used in leishmaniasis chemotherapy stimulates the investigation of new molecules and drug targets for antileishmanial therapy. Methods The activity of 3beta,6beta,16beta-trihydroxylup-20(29)-ene was evaluated against Leishmania (L.) amazonensis by determining the cytotoxicity of the compound on murine peritoneal macrophages, as well as its effects on parasite survival inside host cells. To evaluate the effect of this compound on intracellular amastigotes, cultures of infected macrophages were treated for 24, 48 and 96 h and the percentage of infected macrophages and the number of intracellular parasites was scored using light microscopy. Results Lupane showed significant activity against the intracellular amastigotes of L. (L.) amazonensis. The treatment with 109 μM for 96 h reduced in 80 % the survival index of parasites in BALB/c peritoneal macrophages. At this concentration, the triterpene caused no cytotoxic effects against mouse peritoneal macrophages. Ultrastructural analyses of L. (L.) amazonensis intracellular amastigotes showed that lupane induced some morphological changes in parasites, such as cytosolic vacuolization, lipid body formation and mitochondrial swelling. Bioinformatic analyses through molecular docking suggest that this lupane has high-affinity binding with DNA topoisomerase. Conclusion Taken together, our results have showed that the lupane triterpene from C. leprosum interferes with L. (L.) amazonensis amastigote replication and survival inside vertebrate host cells and bioinformatics analyses strongly indicate that this molecule may be a potential inhibitor of topoisomerase IB. Moreover, this study opens major prospects for the development of novel chemotherapeutic agents with leishmanicidal activity.

To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro.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.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.The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.

This paper presents a novel serine protease (SP) isolated fromBothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated fromBothrops pirajaisnake venom.

In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prND85) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prND85. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prND85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prND85 in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.

Snakebites are a frequently neglected public health issue in tropical and subtropical countries. According to the World Health Organization, 5 million people are bitten annually including up to 2.5 million envenomations. Treatment with antivenom serum remains the only specific therapy for snakebite envenomation. However, it is heterologous and therefore liable to cause adverse reactions, such as early anaphylactic, pyrogenic and delayed reactions. In order to develop alternatives to the current therapy, researchers have been looking for natural products and plant extracts with antimyotoxic, anti-hemorrhagic and anti-inflammatory properties. Especially due to the role the physiopathological processes triggered by snake toxins, play in paralysis, bleeding disorders, kidney failure and tissue damage. Considering the fact that studies involving snake toxins and specific inhibitors, particularly on a molecular level, are the main key to understand neutralization mechanisms and to propose models or prototypes for an alternative therapy, this article presents efforts made by the scientific community in order to produce validated data regarding 87 compounds and plant extracts obtained from 79 species. These plants, which belong to 63 genera and 40 families, have been used by traditional medicine as alternatives or complements to the current serum therapy.

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