Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

Background: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom’s, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

Estudo da ação de inibidores peptídicos de enzimas trombina-símiles na coagulação sanguínea humana / Study of the action of thrombin-like enzymes peptides inhibitors in human blood coagulation

Ophidism is considered the most important cause of accidents by venomous animals around the world. Botropic venom is a highly complex mixture of proteins, peptides and enzymes that, when interacting with human proteins, triggers several local symptoms, such as edema, ecchymosis, pain, blisters and, in more severe cases, necrosis and limb amputation. It also causes systemic symptoms, such as blood incoagulability. The metalloproteases (SVMPs) and the serine proteases (SVSPs) are the major components of Bothrops jararaca venom and are correlated with local and systemic effects. The treatment for poisoning indicated by the Ministry of Health is the administration of botropic antivenom, which despite being highly effective, has certain limitations, such as the partial neutralization of SVSPs. Among the strategies that aim to improve the antivenom and, thus, the treatment of envenomation, is the search for selective inhibitors for SVSPs, which can act simultaneously to the antivenom. Considering the similarity of the catalytic triad between human serine proteases and SVSPs, and since inertia against human serine proteases is a prerequisite for the association of any molecule with antivenom, the present project aimed at the “Study of the action of thrombin-like enzymes peptide inhibitors in human blood coagulation”. The peptide inhibitors, selective for the inhibition of SVSPs, were designed based on a substrate hydrolyzed by these enzymes from Bothrops jararaca venom and showed different inhibition efficacy. In silico analyzes suggest that the inhibitors bound in regions distant from the catalytic triad (His, Asp and Ser) of batroxobin, a thrombin-like enzyme used as a model in studies of enzyme/peptide inhibitor interaction.

O ofidismo é considerado a causa mais importante de acidentes por animais peçonhentos em todo o mundo. O veneno botrópico é uma mistura altamente complexa de proteínas, peptídeos e enzimas que, quando interagem com as proteínas humanas, desencadeiam vários sintomas locais, como edema, equimose, dor, bolhas e, em casos mais graves, necrose e amputação do membro; e sistêmicos, como a incoagulabilidade sanguínea. As metaloproteinases (SVMPs) e as serinoproteinases (SVSPs) são os componentes majoritários do veneno de Bothrops jararaca e estão correlacionadas com os efeitos locais e sistêmicos. O tratamento para o envenenamento indicado pelo Ministério da Saúde é a administração do antiveneno botrópico, que apesar de ser altamente eficaz, apresenta certas limitações, como a parcial neutralização das SVSPs. Dentre as estratégias que visam à melhoria do antiveneno e, assim, o tratamento, está a pesquisa por inibidores seletivos de SVSPs, que possam atuar de forma sinérgica ao antiveneno. Considerando a similaridade da tríade catalítica entre serinoproteinases humanas e as SVSPs, e visto que a inércia contra serinoproteinases humanas é um pré-requisito para a associação de qualquer molécula ao antiveneno, o presente projeto objetivou o “Estudo da ação de inibidores peptídicos de enzimas trombina-símiles na coagulação sanguínea humana”. Os inibidores peptídicos, seletivos quanto à inibição de SVSPs, foram desenhados com base em substrato hidrolisado por estas enzimas do veneno de Bothrops jararaca, e apresentaram diferentes eficácias de inibição. Análises in silico sugerem que os inibidores se ligaram em regiões distantes da tríade catalítica (His, Asp e Ser) da batroxobina, uma enzima trombina-símile usada como modelo em estudos de interação da enzima/inibidor peptídico.