RESUMO

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.

RESUMO

Snakebite accidents are a serious public health problem neglected in tropical countries. In Brazil, the Northern Region has the largest number of cases and snakes from Lachesis genus are responsible for the most serious accidents and the highest number of deaths. Victims of Lachesis envenoming have bite site damage, bleeding and, in severe cases, hypotension. Proteomic and transcriptomic studies report the presence of bradykinin potentiating peptide (BPPs) in L. muta venom, however, there are no data in the literature describing their purifications and identifications. The objective of this work is to identify and characterize angiotensin-converting enzyme inhibitors in the low molecular weight portion of Lachesis muta venom. For this lyophilized L. muta venom was dissolved in ammonium acetate buffer and filtered by centrifugation for size fractionating, using a 10 kDa centrifugal filter unit. The low molecular weight fraction was then chromatographed by RP-HPLC using a C18 column. Twelve fractions were collected and four were tested in fluorimetric assays using ACE as an enzyme and Abz-FRK(Dnp)P-OH as substrate. These fractions present peaks with retention time similar to the peaks that contained BPPs of other snake venoms. In addition, four fractions presented 100% inhibition of the catalytic activity of ACE over the FRET substrate. Based on these results, and due to other data in the literature describing the presence of BPPs in L. muta venom, is possible that it may contain BPPs. For confirmation of this hypothesis, the next step will be to perform analysis by mass spectrometry aiming to determine the primary sequences of the peptides present in each fraction.

Os acidentes ofídicos são um grave problema de saúde pública negligenciado em países tropicais. No Brasil, a Região Norte apresenta o maior número de casos e as serpentes do gênero Lachesis são responsáveis pelos acidentes mais graves e com maior número de óbitos. Vítimas do envenenamento por Lachesis apresentam danos no local da picada, hemorragias e em casos graves apresentam hipotensão arterial. Estudos proteômicos e trancriptômicos descrevem a presença de peptídeos potenciadores de bradicinina (BPPs) no veneno de Lachesis muta, porém não há dados na literatura descrevendo o isolamento e/ou suas identificações. O objetivo deste trabalho é identificar e caracterizar inibidores da enzima conversora de angiotensina (ECA) na porção de baixa massa molecular do veneno de Lachesis muta. Para isso o veneno liofilizado de Lachesis muta foi dissolvido em tampão acetato de amônio e filtrado por centrifugação para o fracionamento de tamanho, usando uma membrana de corte de 10 kDa. A porção de baixa massa molecular foi fracionada por RP-HPLC usando uma coluna C-18. Doze frações foram coletadas e quatro foram testadas em ensaios fluorimétricos utilizando a ECA como enzima e o Abz-FRK(Dnp)P-OH como substrato. Das quatro frações selecionadas, duas se destacaram devido à alta intensidade dos picos, um em 13,65 minutos de retenção e outro em 14 minutos de retenção, com 44% e 45% concentração de solução B, respectivamente. As frações de interesse apresentam picos eluídos nos mesmos tempos de retenção que os BPPs de outros venenos de serpentes, sugerindo, juntamente com dados da literatura, que eles podem conter BPPs semelhantes. As quatro frações inibiram em 100% a atividade catalítica da ECA sobre o substrato Abz-FRK(Dnp)P-OH. Por essas razões e devido outros dados na literatura descreverem a presença BPPs no veneno de L. muta, acreditamos que as quatro frações selecionadas contenham BPPs. Para a confirmação o próximo passo será realizar a análise por espectrometria de massas.

RESUMO

We have previously described a 25mer anti-hypertensive peptide, previously named TsHpt-I (Tityus serrulatus Hypotensin-I), now Ts14, as an agonist of B2 kinin receptor. Bradykinin is known to play physiological roles in angiogenic, inflammatory, and fibrogenic processes, mostly mediated by B2 receptor. Therefore, we investigated whether Ts14 could modulate key events (neovascularization, inflammatory cell recruitment, and extracellular matrix deposition) of the fibrovascular tissue, induced by polyether-polyurethane sponge implants in mice. Sponges were implanted in the dorsum of 7-week-old C57Bl/6 male mice that received daily intrasponge treatment with Ts14 (27.25µg/sponge/day in 10µL PBS) or vehicle (10µL PBS/sponge/day) and were assessed on day 7 after surgery. Hemoglobin content, blood flow (laser Doppler perfusion imaging), and VEGF levels in the implants, used as indices of vascularization, indicated that Ts14 enhanced angiogenesis in implants relative to the PBS-treated group. Interestingly, Ts14 reduced TNF-α levels and neutrophil infiltration, although stimulated macrophage infiltration into implants, as determined by myeloperoxidase (MPO) and N-acetyl-ß-d-glucosaminidase (NAG) enzyme activities, respectively. Regarding the fibrogenic component (soluble collagen content and Sirius-red histological staining), we observed that Ts14 inhibited collagen deposition in the implants. Overall, our results suggest that Ts14 exerts proangiogenic, anti-inflammatory, and anti-fibrogenic activities. These effects may indicate a therapeutical potential of this peptide in conditions where angiogenesis, inflammation, and fibrogenesis contribute to disease progression and chronicity.

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BACKGROUND: The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. METHODS: The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. RESULTS: The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. CONCLUSION: We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. GENERAL SIGNIFICANCE: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

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RESUMO

Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 µM) significantly increased BK-induced contractions (BK; 1 µM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 µM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 µg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 µg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 µg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated.

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Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure-activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs - including BPP-10c - are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.(AU)

Assuntos

RESUMO

Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure-activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs - including BPP-10c - are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 mol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

RESUMO

Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure-activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs - including BPP-10c - are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 mol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

RESUMO

O heptapeptídeo BPP7a, p-Glu1Asp2Gly3Pro4Ile5Pro6Pro7, forma um complexo de associaçãocom a β-ciclodextrina na razão molar 1:1. O peptídeo e a sua forma complexa foram caracterizados por dicroísmo circular (CD) e titulação calorimétrica (ITC), as quais sugerem uma interação muito fraca entre a β-ciclodextrina e o peptídeo. Espectros de ressonância magnética nuclear (NMR) de 1H a 400 e 600 MHz foram obtidos para o peptídeo puro e para o complexo com β-ciclodextrina e com estes foi possível a atribuição de todos os sinais de ressonância de hidrogênio do peptídeo. Experimentos de espectroscopia ordenada de difusão de alta resolução (HR-DOSY) foram conduzidos a fim de se confirmar a associação entre o BPP7a e a β-ciclodextrina, além de se verificar a quebra dos agregados moleculares do BPP7a devida a associação. A atividade antihipertensiva do complexo BPP7a/β-ciclodextrina foi avaliada em ratos naturalmente hipertensivos (SHR), mostrando resultados melhores do que os do peptídeo BPP7a puro.

The BPP7a heptapeptide, p-Glu1Asp2Gly3Pro4Ile5Pro6Pro7, forms an association complexwith β-cyclodextrin in a 1:1 molar ratio. The peptide and its complex were characterized bycircular dichroism (CD) and isothermal titration calorimetry (ITC), which showed a very weak interaction between the β-cyclodextrin and the peptide. Assignments of all hydrogen resonances of the peptide alone and as a complex were made using 1H nuclear magnetic resonance (NMR)experiments at 400 and 600 MHz. High resolution diffusion ordered spectroscopy (HR-DOSY) experiments were carried out to establish the self-aggregation state of BPP7a. It was also shown that the β-cyclodextrin breaks the molecular clusters leading to complex formation. In addition,the anti-hypertensive activity of the BPP7a/β-cyclodextrin complex was evaluated in spontaneous hypertensive rats (SHR), showing increased activity compared to that of pure BPP7a.

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RESUMO

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

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