Rational design of inhibitors of the mitochondrial enzyme alternative oxidase (AOX) with fungicide activity against Moniliophthora perniciosa, causal agent of the witches' broom disease of cocoa

Author(s)

Mario Ramos de Oliveira Barsottini

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-02-18

Examining board members

Gonçalo Amarante Guimarães Pereira; Celso Eduardo Benedetti; Juliana Ferreira de Oliveira; Luis Eduardo Soares Netto; Paulo Henrique Conaggin Godoi

• Advisor: Gonçalo Amarante Guimarães Pereira

Abstract

The cacao tree seeds (Theobroma cacao) are the raw material used in the chocolate industry. However, fungal diseases endanger the world cocoa production and generate losses of 30%. In Brazil, the most important cocoa pathogen is Moniliophthora perniciosa, which devastated farms in the state of Bahia after an outbreak in 1989. In addition to economic losses, M. perniciosa caused severe social and ecological impacts, such as unemployment, rural exodus and uncontrolled urban growth. M. perniciosa is the causal agent of the witches¿ broom disease (WBD) of cocoa, which is currently controlled with tolerant cacao clones and agronomic management practices. Nonetheless, it is not possible to eradicate WBD, which greatly limits the potential for recovery of Brazilian cacao farms. It has been shown that the mitochondrial enzyme alternative oxidase (AOX) is a potential target for the development of new fungicides against M. perniciosa. AOX is a membrane-bound protein associated with the internal membrane in the mitochondria and a terminal oxidase. As such, AOX creates a branching point in the electron transport chain involved in the oxidative phosphorylation, which reduces ATP synthesis, but also confers metabolic plasticity to the cell to resist against a range of abiotic and biotic stresses. In M. perniciosa, MpAOX acts as an escape mechanism against the cacao defense response, as well as fungicides widely used in agriculture. Therefore, the main goal of this work was to generate relevant information for the directed development of MpAOX inhibitors with potential antifungal activity, which are presented in two chapters. The first chapter presents the development of a new experimental model based on the Pichia pastoris yeast for the functional characterization of N-phenylbenzamide derivatives (NPD). Oxygen consumption measurements and growth assays allowed the identification of at least one selective AOX inhibitor with antifungal activity in vitro and in planta. The second chapter addresses the heterologous expression, purification and functional characterization of MpAOX. Ascofuranone and structurally-related AOX inhibitors were subjected to dose-response assays, which led to the identification of key requirements for favorable ligand interaction with MpAOX and other isoforms. Collectively, those results will assist in the targeted development of a novel antifungal agent targeting M. perniciosa and other fungal pathogens dependent on AOX, which will contribute to Brazil's scientific and technological advancement in strategic areas such as agriculture and food security (AU)

• FAPESP's process: 14/15339-6 - Characterization of the inhibition of the enzyme alternative oxidase (AOX) by aromatic amides and development of high-throughput screening platform to search for novel inhibitors of the AOX from the fungus Moniliophthora perniciosa
• Grantee: Mario Ramos de Oliveira Barsottini
• Support Opportunities: Scholarships in Brazil - Doctorate