Isolation and functional and structural characterization of antitumoral compounds with low molecular mass of Bothrops jararacussu snake venom

Author(s)

Gabriel Neves Cezarette

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-06-23

Examining board members

Suely Vilela; Antonio Eduardo Miller Crotti; Elisa Maria de Sousa Russo

• Advisor: Suely Vilela

Abstract

Snake venoms are complex mixtures of biological active compounds, among them, still little explored, the low molecular weight compounds such as peptides, organic components and lipids. These molecules have diversified pharmacological activity, and are showing increasing scientific interest about their potential to be an anticancer agent. Although there are several reports of peptides from classes with a well-known antitumor activity in this venom, these have never been effectively isolated. In this way, the present study aimed to isolate the low molecular weight of the Bothrops jararacussu snake venom, which presents an antitumor action, in an approach that considers direct pharmacological or mediated by biological pathways, and are included in the most current aspects of understanding tumorigenesis and its therapy. The bioprospecting strategy for these compounds used different tumor cell lines, such as cellular hepatocarcinoma (HepG2), prostate carcinoma (DU-145), lung carcinoma (A549), and breast adenocarcinoma (MDA-MB-231), for the identification of cytotoxic activities, and non-cytotoxic in vitro tumorigenesis inhibition mechanisms, such as the modulation of cell migration and proliferation. The crude venom was ultrafiltered in specific membranes, isolating the low molecular weight compounds, and this pool was fractionated by molecular exclusion chromatography. The bioactive molecules of these fractions were identified through in vitro biological assays, and purified by reversed-phase chromatography. The results of this project identified four compounds with cytotoxic activity, named Js II-I; Js III-I; Js IV-II; and Js VI-I, being able to induce an increase in apoptosis and/or autophagy rates, in addition to reducing the survival of cells exposed to its treatment. Js III-I, the compound that presented the highest cytotoxic potential, had its mechanisms of action investigated, where it was shown to be able to induce the apoptosis process through the activation of the BAX, BCL-2 and FAS gene pathways, and activation of the enzymes of the intrinsic pathway. of apoptosis (Caspase-3 and -9). The bioactive compounds were structurally characterized by mass spectrometry and nuclear magnetic ressonance, and their toxicity to normal human cells was investigated by MTT assay in peripheral blood mononuclear cells and by hemolytic activity, being observed no toxic action for any of these compounds. The results described in this work have an unprecedented character, as they are the first reports of the isolation and functional characterization of low molecular weight compounds with antitumor action in the venom of the snake B. jararacussu, and contribute to the identification of four new compounds with potential for be used in studies of the development of new drugs. (AU)

• FAPESP's process: 20/03674-6 - Isolation and functional and structural characterization of antitumor peptides from the Bothrops jararacussu snake venom
• Grantee: Gabriel Neves Cezarette
• Support Opportunities: Scholarships in Brazil - Master