A Kunitz-type peptide from Dendroaspis polylepis venom as a simultaneous inhibitor of serine and cysteine proteases

Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. Methods: The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. Results: The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. Conclusion: This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.(AU)

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.(AU)

Eficácia do soro antibotrópico produzido no Instituto Butantan: neutralização de diferentes classes de enzimas proteolíticas do veneno total da Bothrops jararaca / Efficacy of bothropic antivenom produced by Butantan Institute: neutralization of different classes of proteolytic enzymes from the venom of Bothrops jararaca

O envenenamento ofídico representa uma questão de saúde para vários países do mundoe alguns fatores importantes devem ser levados em consideração a fim de minimizareste problema como, por exemplo, atendimento médico de qualidade e um melhor registro de acidentes. O gênero Bothrops é considerado o mais importante nos casos deenvenenamento ofídico no Brasil e, como outras serpentes, seu veneno é uma mistura complexa de componentes que agem em diferentes alvos na presa/vítima. Considerandoque as enzimas proteolíticas das classes das metalopeptidases e as serinopeptidases (cerca de 65 % da composição do veneno) são as principais toxinas do veneno da B.jararaca, as mesmas foram escolhidas no presente estudo para se determinar o nível de neutralização das suas atividades pelo soro produzido pelo Instituto Butantan, o qual éamplamente utilizado no Brasil. Para isso dois substratos FRETs foram selecionados, a partir de uma biblioteca de peptídeos, que se comportaram como alvos seletivos para as serino- e metalopeptidases. Desta maneira, observamos que o soro é ineficiente no bloqueio da atividade de duas serinopeptidases e, este fato nos levou à busca dos motivos pelos quais o soro antibotrópico comercial estaria apresentando esta falha. Assim, estas duas serinopeptidases ainda inéditas no veneno de Bothrops jararaca e não neutralizadas pelo soro comercial foram purificadas e identificadas e verificamos que a razão na falha do soro comercial em bloquear estas atividades é ausência de anticorpos específicos contra estas enzimas já que as mesmas não eram reconhecidas pelo soro no ensaio de western blot. Assim, nossos resultados indicam uma possível falha na composição do soro antibotrópico, portanto fomos levados a estudar mais profundamente o papel destas serinopeptidases e buscamos novos substratos para estasenzimas...