In vitro comparative metabolomics evaluation of a drug candidate for leishmaniasis treatment

Author(s)

Gisele André Baptista Canuto

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2016-06-03

Examining board members

Marina Franco Maggi Tavares; Ana Valéria Colnaghi Simionato Cantu; Cassiana Seimi Nomura; Pedro Vitoriano de Oliveira; Daniel Carvalho Pimenta

• Advisor: Marina Franco Maggi Tavares

Abstract

Leishmaniasis is a neglected disease caused by Leishmania parasites, which occurs in more than 90 countries worldwide, affecting millions of people. The disease is treatable but not curable, and the commonly used therapeutic arsenal is limited and consists in the use of highly toxic drugs. The search for new therapies has been encouraged and the use of active compounds, isolated from natural products, such as plants, has been proven effective. For future use of these active compounds in the treatment, it is necessary to understand its mechanism of action. \"Omics\" sciences, specifically metabolomics, which is the comparative analysis of altered metabolites in a biological system after external intervention, has been widely applied for this purpose. The metabolomic approach is multidisciplinary and the analyzes are performed using modern analytical platforms, such as separation techniques coupled to mass spectrometry (MS). Data treatment are performed with advanced statistical tools and altered metabolites are correlated with metabolic pathways. In this work, the mechanism of action, in Leishmania infantum, of an active compound (methyldehydrodieugenol B), isolated from Nectandra leucantha plant, was evaluated by global metabolomics approach with multiplatform analysis (gas and reversed phase liquid chromatography, GC-MS and RPLC-MS, respectively). Optimizations of sample preparation, extraction and derivatization for GC-MS to obtain the maximum number of metabolites in the samples under consideration were performed. L. infantum promastigotes were grown in culture medium and after 72 h they were treated with metildihidrodieugenol B (at a concentration of 58.18 µg x mL-1); after 48 h of treatment, enzyme activity was quenched, cells washed and frozen for further analysis. Methanol:water (1:1) and 100% methanol are used as solvent extractor to assess intracellular metabolites for analysis by GC-MS and RPLC-MS, respectively. Derivatization with O-methoxyamine in pyridine (15 mg x mL-1) at room temperature for 90 min, followed by silylation with BSTFA + 1% TMCS for 30 min at 40°C was performed to make volatile and stable metabolites for analysis by GC- MS. The results of both analytical platforms showed significant differences between treated and non-treated samples with the active substance, and altered metabolites with statistical significance were correlated with Leishmania metabolism. Different amino acids, fatty acids, carbohydrates, and glycerolipids phospholipids were found, mostly decreased with treatment. Due to the complexity of the parasite metabolism and the great diversity of altered metabolites, a multi-target mechanism can be assigned to the methyldehydrodieugenol B. Changes in the metabolism of glycolysis and gluconeogenesis are highlighted, indicating changes in the cell energy sources. Changes in the lipid composition of the parasite plasma membrane were also observed, suggesting variations in its flexibility and fluidity. The results are preliminary, but very important to take the first step into elucidation of the action of this substance in the treatment of leishmaniasis, aiming the search for new pharmaceutical therapies. (AU)

• FAPESP's process: 12/04601-6 - IN VITRO COMPARATIVE METABOLOMIC EVALUATION OF CANDIDATE DRUGS FOR LEISHMANIASIS TREATMENT
• Grantee: Gisele André Baptista Canuto
• Support Opportunities: Scholarships in Brazil - Doctorate