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MOLECULAR BIOLOGY

Allies for an antidotal serum

Medicinal plants may reduce local lesions caused by a jararaca snakebite

Bothrops insularis or Golden Lancehead, native to Queimada Grande Island on the coast of São Paulo State

Eduardo CesarBothrops insularis or Golden Lancehead, native to Queimada Grande Island on the coast of São Paulo StateEduardo Cesar

Each year in Brazil, around 30,000 people become victims of snakebite, according to data from the Ministry of Health. The principal villains on the list of perpetrators, responsible for some 80% of cases, are jararacas, snakes of the genus Bothrops, which are found in every region of Brazil. The medical advice in such cases is clear: the victim should be given the antidotal serum immediately. “The serum works systemically. It can minimize blood-clotting disturbances and renal failure and prevent death, but in the case of jararacas, it does not protect against local lesions, such as wounds and necrosis, that can lead to the amputation of an arm or a leg,” says biologist Carlos Fernandes, from the Structural Molecular Biology Laboratory (LBME) at São Paulo State University (Unesp), Botucatu campus. Searching for alternatives, he showed that plants used by traditional and indigenous communities are effective in treating local lesions, publishing his most recent findings in the journal PLOS One in July 2015. “We are hoping that an ointment, for example, might complement the effect of the serum in the near future.”

Prior to showing the effect of these plants, the Unesp group needed to solve an enigma involving jararaca venom. In the 1980s, studies conducted in other countries indicated that, in jararaca venom, phospholipase A2 proteins, which are commonly found in the venom of many snakes, exhibit structural modifications that intensify its local effects.

Fernandes and colleagues employed crystallography, the principal technique used to understand the three-dimensional structure of proteins, and identified two amino acids that occupy different positions in the modified phospholipases. They also showed that the two types of phospholipases act differently on muscle cells. Whereas traditional proteins cause cell rupture, the modified ones initially cause less damage. They perforate the cell membrane and create an imbalance in the flow of ions that leads to ostensibly slower cell death. In combination, however, the two forms accelerate the formation and increase the extent of the wound. “We initially set out to understand the spatial organization of the amino acids, and then to describe the mechanisms by which the cell membranes are damaged,” says Fernandes, who published the findings in the journal Biochimica et Biophysica Acta in 2013 and 2014. “These data were needed so that we could look for a compound that can complement serum therapy,” he says.

Protective effect
With that objective in mind, the Unesp group tested molecules present in three species of medicinal plants: aristolochic acid (found in a plant native to the Atlantic Forest known as jarrinha or papo-de-peru), rosmarinic acid (from the cordia plant, native to the same area) and caffeic acid (abundant in the leaves of the boldo-baiano or assa-peixe, plants native to Africa). In the laboratory, the researchers first examined what happens to the muscles of mice in contact with only jararaca venom, and then what occurs after adding each of the compounds. “In the first situation, the muscle is damaged and loses the ability to contract. In the second, with either substance, the muscle is preserved,” explains Marcos Fontes, coordinator of the LBME.

The next step is to seek out partnerships with institutions with recognized competence in drug testing, such as the Butantan Institute or the Oswaldo Cruz Foundation, to begin preclinical trials that might lead to the manufacture of a plaster or ointment that can be applied locally. Catarina Teixeira, from the Pharmacology Laboratory at Butantan, recently began researching the antidotal effect of several plant-derived substances, and she considers the diversity of Brazilian flora to be a valuable arsenal for confronting this public health problem. In a study conducted in her own laboratory, Mônica Kadri, of the Federal University of Mato Grosso do Sul, confirmed that the extract from the bark of the ipê or trumpet tree (Tabebuia aurea), commonly found in the Pantanal wetland, has anti-inflammatory and scarring effects and can minimize the effect of jararaca venom at the site of the snakebite.

“The attempt to combine plant-based treatment with serum therapy is an old line of research, but we need greater coordination between researchers from different specialties to make it a reality,” Teixeira comments. The immediate challenge for research groups is to verify whether the effect observed in vitro is maintained in vivo. “What we want,” says Fontes, “is that one day, snakebite victims can immediately use an ointment applied at the site of the wound and try to obtain the serum.”

Projects
1. Structural and functional studies of native, recombinant and complexed proteins, from snake venom, with vegetable inhibitors (nº 2012/06502-5); Grant Mechanism: Regular Research Grant; Principal Investigator: Marcos Roberto de Mattos Fontes (Unesp); Investment: R$743,409.00.
2. Structural studies with neurotoxic phospholipases A2 (nº 2013/17864-8); Grant Mechanism: Scholarships in Brazil – Postdoctoral; Principal Investigator: Marcos Roberto de Mattos Fontes (Unesp); Recipient: Carlos Alexandre Henrique Fernandes (Unesp); Investment: R$168,748.00.
3. National Institute of Science and Technology on Toxins (nº 2008/57898-0); Grant Mechanism: Thematic Project-INCT; Principal Investigator: Osvaldo Augusto Brazil Esteves Sant’anna; Investment: R$3,974,330.00 (FAPESP/CNPq/CAPES).

Scientific articles
FERNANDES, C. A. H. et al. Structural basis for the inhibition of a phospholipase A2-like toxin by caffeic and aristolochic acids. PLOS One. V. 10, No. 7, e0133370. July 20, 2015.
FERNANDES, C. A. H. et al. A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A2-like proteins from viperid snake venoms. Biochimica et Biophysica Acta. V. 1844, No. 12, p. 2265-76. September 30, 2014.
FERNANDES, C. A. H. et al. Structural bases for a complete myotoxic mechanism: crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom. Biochimica et Biophysica Acta. V. 1834, No. 12, p. 2772-81. December 2013.

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