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Structural and pharmacological characterization of toxins isolated from Bothrops bilineatus smaragdinus (forest viper) venom with potential modulatory activity of the motor synaptic release and cardiovascular functions

Grant number: 20/04287-6
Support type:Regular Research Grants
Duration: August 01, 2021 - July 31, 2023
Field of knowledge:Biological Sciences - Pharmacology - Toxicology
Principal researcher:Rafael Stuani Floriano
Grantee:Rafael Stuani Floriano
Home Institution: Pró-Reitoria de Pesquisa e Pós-Graduação. Universidade do Oeste Paulista (UNOESTE). Presidente Prudente , SP, Brazil
Assoc. researchers: Bruno Lomonte ; Edward Gerard Rowan ; Francis Lopes Pacagnelli ; Stephen Hyslop


Envenomation by lancehead snakes (genus Bothrops) is characterized by cardiovascular effects (coagulopathy, systemic hemorrhage, hypotension/shock and acute kidney injury) mediated by enzymes such as L-amino acid oxidases, C-type lectins, serine proteinases, metalloproteinases and phospholipases A2 (PLA2), with the some of the latter acting as b-neurotoxins to cause presynaptic neuromuscular blockade. The cardiovascular effects of envenomation by Bothrops spp. are considered the principal cause of death, with little peripheral neurotoxicity. The toxinology of Bothrops bilineatus smaragdinus venom has not been systematically studied. In this project, we intend to purify and structurally and pharmacologically characterize presynaptic neurotoxins from this venom and examine their ability to modulate cardiovascular functions in vitro. Initially, the venom will be fractionated by gel filtration to obtain fraction P-3 that will then be purified using RP-HPLC; the resulting toxins will be screened for neurotoxicity in mouse isolated phrenic nerve-diaphragm (PND) preparations in vitro. The toxins will also be assessed for enzymatic and cytotoxic activities followed by molecular identification through sequencing with mass spectrometry. The mechanisms involved in the neuromuscular effects of the principal neurotoxins will be examined using a variety of electrophysiological approaches, including intracellular (evoked and spontaneous end-plate potentials) and extracellular (compound action potentials) recordings in mouse nerve-muscle preparations, as well as ion current (whole cell patch-clamp) recordings in primary cultures of ganglion neurons. The cardiac (rat isolated atria) and vascular (rat isolated thoracic aorta) actions of the toxins will also be assessed in vitro. The results of this work will contribute to our understanding of the mechanisms involved in envenomation by this specie and allow the characterization of toxins with a potential modulatory action of neuronal and cardiovascular functions. (AU)