Purification and characterization of peptides present in African snake venoms: searching for inhibitors of medical importance peptidases

As peptidases desempenham um papel importante para o bom funcionamento fisiológico dos mais diversos organismos. Quando desreguladas, estão associadas a patologias, por exemplo, a atividade exacerbada da elastase-1 está relacionada à pancreatite, e, da catepsina L, está envolvida na metástase de tumores. Portanto estas peptidases se tornaram potenciais alvos terapêuticos, no que se refere à procura por inibidores. O veneno de serpentes é composto por misturas complexas de moléculas que podem apresentar as mais derivadas funções, inclusive a inibição de enzimas. Tendo isto em vista, este trabalho teve como objetivo, purificar, caracterizar e estudar in vitro e in silico novos inibidores presentes nas porções de baixa massa molecular dos venenos de três gêneros de serpentes africanas, Naja, Dendroaspis e Bitis. Para isso, as porções de baixa massa molecular (<10 kDa) de cada veneno foram submetidas a uma cromatografia por RP-HPLC usando uma coluna C18. As frações foram coletadas e um screening, utilizando substratos FRET, foi feito para verificação da inibição sobre as peptidases estudadas neste trabalho. A identificação das frações de interesse foi realizada por espectrometria de massas, dentre as sequências obtidas, moléculas da classe das three-finger-toxins foram identificadas, e, pela primeira vez descritas como inibidores de peptidases. Outras moléculas, da classe dos Kunitz, que foram anteriormente descritas como bloqueadores de canais de Na+ e K+, também foram identificadas. O peptídeo DPSA8 (do veneno de Dendroaspis polylepis) se destacou por apresentar inibição semelhante da catepsina L e da elastase-1 no teste de screening. Este peptídeo, classificado como Kunitz, foi caracterizado como um inibidor acompetitivo para a elastase-1, com um Ki de 8 &#181M. A análise por ancoramento (ZDOCK) indicou modelos de interação deste peptídeo com a elastase-1, dentre estes modelos foram mostradas interações no sítio catalítico e em outras regiões da enzima. Utilizando estes modelos de interação, dois peptídeos foram desenhados para serem estudados quanto à possível inibição da elastase-1 e da catepsina L. O primeiro não apresentou atividade inibitória, entretanto, o segundo, um peptídeo cíclico nomeado PEP2 (contendo 30 resíduos de aminoácido), mostrou ser um inibidor competitivo, com Ki de 1,96 &#181;M sobre a catepsina L. Além disso, foi realizada a análise de ancoramento dos BPPs, do veneno de Bitis spp, usando a ECA (enzima conversora de angiotensina I) como receptor. Estes resultados mostraram uma alta relação dos scores obtidos nos estudos in silico com os Kis obtidos e já publicados. Este trabalho descreve algumas toxinas de baixa massa molecular com a função inibitória de peptidases, isso pode contribuir para um entendimento maior do processo de envenenamento. Em adição, foi empregado o método de ancoramento molecular para o desenho de novas moléculas inibidoras, resultando no PEP2, um peptídeo capaz de inibir a atividade da catepsina L com uma baixa constante de inibição. Mostrando mais uma vez que esta metodologia pode contribuir para a procura de novas entidades biotecnológicas.Peptidases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated to pathologies, for example, the exacerbated activity of elastase-1 is related to pancreatitis, and, for the cathepsin L, it is involved in the tumors metastasis. Therefore these peptidases have become potential therapeutic targets regarding the search for inhibitors. The venoms of snakes are composed by complex mixtures of molecules that can feature diverse functions, including the inhibition of enzymes. Considering this, the present works objective was to purify, characterize and to study in vitro and in silico new inhibitors that are present in the low molecular weight fraction of the venoms from three African snakes genera, Naja, Dendroaspis and Bitis. For that, the low molecular weight fraction from each venom (<10 kDa) were submitted to RP-HPLC chromatographic fractioning, using a C-18 column. The fractions were them collected, and utilizing fluorescent substrates, the screening test was performed to verify the inhibition by the fractions over the peptidases studied in this work. The identification of the fractions of interest was carried out by mass spectrometry, among them, molecules from the three-finger-toxins class was identified, and, for the first time, described as peptidase inhibitors. Other molecules from Kunitz class, that were previously described as Na+ and K+ channel blockers, but not as peptidase inhibitors, were also identified. The peptide DPSA8P (from Dendroaspis polylepis venom) stood out due to the fact that it had similar cathepsin L and elastase-1 inhibition on the screening step. This peptide, classified as a Kunitz one, was characterized as an incompetitive inhibitor for elastase-1, presenting a Ki of 8 &#181;M. The docking (ZDOCK) analysis indicated interaction models of this peptide with elastase-1, in the catalytic site and in other regions of this enzyme. Using these interaction models, two peptides were designed to be studied as possible inhibitors of both elastase-1 and cathepsin L. The first one did not show inhibitory activity, however, the second, a cyclic peptide named as PEP2, which contains 30 amino acid residues, showed to be a competitive inhibitor, with Ki of 1.96 &#181;M over cathepsin L. Furthermore, we performed re-docking analyses of the BPPs, from the Bitis spp venom, using the ACE (angiotensin converting enzyme) as receptor. These results showed high relation of the scores obtained in the in silico studies with the Kis previously determined. This work describes some low molecular weight toxins with a new function as peptidase inhibitors, and this could contribute to further understand the envenomation process. In addition, the efficiency of the docking technique to design new molecules was shown, aiming at the search molecular docking was used to the design new of peptidase inhibitors, which led to the PEP2, a peptide that is able to inhibit the cathepsin L activity with a low inhibition constant. Showing once again that this methodology may be used for the search of new biotechnological entities

Analysis of the low molecular mass constituents from the venom of four species of the Bitis genus and biological activities.

Na África subsaariana, as serpentes do gênero Bitis são de extrema importância, pois suas vítimas apresentam sintomas como dano local, hemorragia e uma severa hipotensão. Este trabalho identificou moléculas capazes de inibir a atividade da enzima conversora de angiotensina I (ECA) presentes no veneno de quatro serpentes do gênero Bitis. Para isto, as porções de baixa massa molecular desses 4 venenos foram fracionadas em RP-HPLC e as frações com boa inibição sobre a atividade da ECA foram analisadas por espectrometria de massas. Foram identificados 34 oligopeptídeos ricos em prolina (PRO), sendo 8 sintetizados e suas constantes de inibição (Ki) determinadas. Em testes com substratos naturais da ECA, angiotensina I e bradicinina, foi constatada a maior inibição da hidrólise da angiotensina I por quatro PROs. Todos os PROs in vivo reduziram a pressão arterial, e seis deles aumentaram a frequência cardíaca em ratos Wistar. Com isto, conclui-se que existem toxinas no veneno de serpentes do gênero Bitis responsáveis pela hipotensão.In the sub-saharian Africa, snakes from the Bitis genus are of extreme medical importance, since its victims show symptoms as local tissue damage, hemorrhage and a severe hypotension. This work identified molecules that inhibit the angiotensin I converting enzyme (ACE) in the venom of 4 snakes from the Bitis genus. The low molecular portions of the venom of these snakes were fractionated in RP-HPLC and the fractions that efficiently inhibited the ACE activity were analyzed by mass spectrometry. 34 proline-rich oligopeptides were identified, 8 of them synthesized and had their inhibition constants (Ki) determined. In tests using natural substrates of ACE, angiotensin I and bradykinin, the angiotensin I hydrolysis were better inhibited by four PROs. In vivo tests results showed that all PROs decreased the mean arterial pressure and six of them increased the heart rate. Therefore, we can conclude that there are toxins present in the venom of Bitis capable of cause hypotension

Comprehensive Analysis and Biological Characterization of Venom Components from Solitary Scoliid Wasp Campsomeriella annulata annulata

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear &alpha;-helical peptides. Among the components of the first class, the two main peptides, &alpha;-campsomerin (PRLRRLTGLSPLR) and &beta;-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only &alpha;-campsomerin was able to enhance cell viability, while &beta;-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of &beta;-campsomerin, compared to &alpha;-campsomerin. Among the linear &alpha;-helical peptides, annulatin (ISEALKSIIVG-NH2) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities

New Insights into the Hypotensins from <i>Tityus serrulatus</i> Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 μM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice’s naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-

[des-arg(1)]-proctolin: a novel nep-like enzyme inhibitor identified in tityus serrulatus venom

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys801824CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2014/12976-5; 2012/06677-0; 2013/15343-0Invertebrate Neuropeptide Conference 2015 (INC2015)2015MyanmarBaganInstituto Nacional de Ciencia e Tecnologia em Toxinas-Conselho Nacional de Desenvolvimento Cientifico e Tecnologic

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the <i>Tityus serrulatus</i> Venom

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension

Novel Cysteine Protease Inhibitor Derived from the <i>Haementeria vizottoi</i> Leech: Recombinant Expression, Purification, and Characterization

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models

Comparing Traditional and Toxin-Oriented Approaches towards Antivenom Production against <i>Bitis arietans</i> Snake Venom

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom (“traditional approach”) and immunization with selected key toxins isolated from the snake venom (“toxin oriented” approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa