According to the World Health Organization, ophidism is considered a neglected disease. It is estimated around 5 million cases of snakebites per year resulting in more than 100,000 cases of death and 400,000 irreversible damage, including amputations and reduced mobility. In Brazil, most ophidic accidents are caused by snakes of the Bothrops genus. This study aims the heterologous expression of BJ46a, which act as an inhibitor of the hemorrhagic metalloprotease jararhagin. The goal of this study is the evaluation of the efficacy of recombinant inhibitor in first aid application in order to reduce the damage caused by the eventual delay in the conventional treatment by B. jararaca antiophidic serum. The expression of the gene encoding the inhibitor is performed using the methylotrophic yeast Pichia pastoris and the assay will be conducted in the presence of jararhagin to measure its efficiency.
In 2021, our team participated in iGEM by proposing the synthesis of an proteic inhibitor (𝛾PLI) for phospolipase A2, an enzyme responsible for necrosis. This enzyme is present in the venom of the Bothrops jararaca snake. In 2022, we returned with a proposal to further develop and add an inhibitor for hemorrhage, in addition to the previous project experiments idealized.
Ophidic accidents represent a serious public health problem in tropical countries due to their frequency and mortality. There are approximately 3,000 species of snakes in the world, and 10 to 14% of these species are considered poisonous. In Brazil, according to data from the Ministry of Health, 19,000 to 22,000 ophidic accidents occur every year (PINHO; PEREIRA, 2001).
During a 10 years period (2003 to 2013), about 307,770 ophidic accidents were reported in Brazil, and 90% attributed to snakes of the genus Bothrops, which represents the group of jararacas, thus evidencing its great relevance in the medical field (NATIONAL; SANITARY, [s.d.]).
The occurrence of ophidic accidents is, in general, related to climatic factors and the increase in human activity in field work. The age ranges from 15 to 49 years, with males being the most prevalent. As for the site of the bite, the foot and leg are the most affected areas.
According to the World Health Organization (WHO), it is estimated that between 81,000 and 138,000 people die annually worldwide as a result of venomous snakebites, and another 400,000 become permanently disabled or disfigured. In 2020, in Brazil, 31,395 snake accidents were recorded in the official systems of the Ministry of Health, of which 121 led to the death of the victims.
Currently, the treatment indicated for ophidic accidents is the serum produced from large animals, especially horses. The production of this serum goes through several steps, starting with the extraction of the snake venom, followed by the transformation into antigen. Small doses of this antigen are applied to the horse body in order to stimulate the production of antibodies. When there are enough antibodies in the animal blood, its plasma is collected. Finally, the plasma goes through a complex industrial processing to obtain the serum.
Figure 1 - Illustration of the production of conventional antiophidic serum
B. jararaca is one of the snakes most involved in ophidic accidents in Brazil. Predominantly terrestrial, they have nocturnal habits and prefer humid places like forests or cerrado areas (Hartmann et al., 2003).
According to Grazziotin (2004), the body of these snakes is usually dark near the head, and on the back it has triangular designs and other distinctive shapes to camouflage in dry leaves (Figure 2). The diet consists mainly of small rodents, which allows them to grow up to 160 cm. Its viviparous reproduction is seasonal, with birth predicted for the hot and humid summer (ALVES et. al, 2000).
Figure 2 - Bothrops jararaca
Source: Fiocruz
Like other snakes of the Bothrops genus, jararaca venom has high concentrations of metalloproteases, representing about 53% of the composition of botropic venom (CIDADE, 2006), indicating a large role of this class of proteins in the envenomation.
Jararhagin is a metalloprotease present in Bothrops jararaca venom that has a high hemorrhagic effect. By inhibiting platelet aggregation induced by collagen and ristocetin, jararhagin interferes with the blood clotting process, reducing platelet activities and causing consequent hemorrhagic lesions. Furthermore, snake venom metalloprotease also has an inhibitory effect on the adhesion of endothelial cells to collagen-coated plaques, and thus leads to a weakening of blood vessel walls and the collaboration of such bleeding lesions (MOURA-DA-SILVA; BALDO, 2012).
Therefore, analyzing the context of the current production of the antiophidic serum and the effects of the toxins present in the Bothrops jararaca venom, especially jararhagin, it is understood the importance of an alternative way of producing an effective drug with greater control and ease in purification, less time spent in production and the absence of the need to use animals during the process. Thus, we propose the production of BJ46a inhibitor by means of synthetic biology techniques in order to contemplate these alternatives, mentioned above, in a full way.
BJ46a is a natural snake venom inhibitor and is classified within the snake venom metalloproteinase inhibitors (SVMPI). Extracted from the plasma of the snake B. jararaca, this SVMPI has two domains, the cystatin and the histidine-rich domain in its C-terminal portion (BASTOS, 2014). The mechanism of action of BJ46a involves a direct interaction of the inhibitor with the zinc-dependent metalloproteinase, which leads to enzyme inhibition (Valente et al, 2001).
During the development of the project, the following objectives were met: