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Search of inhibitors of typical 2-Cys peroxirredoxin system from eukaryotes: functional and structural evaluation

Grant number: 19/04054-4
Support type:Scholarships in Brazil - Master
Effective date (Start): August 01, 2019
Effective date (End): July 31, 2021
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Ana Laura Pires de Oliveira
Home Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
Associated research grant:13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

The massive production of reactive oxygen and nitrogen species (ROS and RNS) represents one of the main lines of defense of hosts against the attack of pathogenic microorganisms. However, in the course of evolution, microorganisms have developed highly efficient enzymes capable of decomposing ROS and RNS and neutralizing host defenses, such as peroxiredoxins (Prx), which are distinguished by the decomposition of a great variety of hydroperoxides with high specificity and efficiency (k = 10^6 - 10^8 M-1 s-1), using a highly reactive cysteine called the peroxidase cysteine and distributed in the most diverse cellular environments. These enzymes have structural and functional variability and can be divided into three classes: 1-Cys, typical 2-Cys and atypical 2-Cys Prx. Typical 2-Cys Prx are the most abundant in organisms, contributing decisively to the maintenance of cellular homeostasis and are the object of this study. In pathogenic fungi, oxidative/nitrosative stress promoted by defense cells and/or fungicides is used to control infection in the host. However, in immunocompromised individuals, invasive infections and strains resistant to the main fungicides, such as amphotericin B and azoles, represent a serious public health problem. In this context, typical 2-Cys Prx of pathogenic fungi appear to be a very important target for fungicides, however no typical 2-Cys Prx inhibitor has been characterized for these organisms. On the other hand, inhibitors of these enzymes have been described for mammals, among them natural diterpenoids such as Adenantine (Adn) and JDA-202, which are capable of inhibiting human cytosolic 2-Cys isoforms, leading to tumor remission and presenting low toxicity to healthy cells. It is important to note that studies investigating typical 2-Cys Prx inhibitors have never attempted to evaluate comparably with enzymes from other organisms, where there is a possibility that the same molecule could be applied for other purposes such as fighting pathogenic organisms. Our research group has demonstrated that Adn is able to inhibit the typical 2-Cys cytosolic isoforms Tsa1 and Tsa2 from S. cerevisiae, a model organism for biochemical and genetic studies. Investigations carried out within the scope of the project proposed here revealed that Tsa1 shows high identity with cytosolic isoforms of pathogenic yeasts Candida and Cryptococcus and in Rhodotorula mucilaginosa, revealing that this yeast is a good model for the study of these organisms. The objective of this project is to search for natural compounds from the São Paulo coastal biota with inhibitory activity on fungal 2-Cys Prx, using as a Tsa1 of S. cerevisiae model in a comparative manner with Prx2, the most abundant isoform of typical 2-Cys Prx of humans. This yeast will also be used for in vivo assays of the fungicidal potential of the compounds identified as inhibitors of Tsa1 and also of inhibitors + fungicides widely used in clinical treatments. In addition, the hydroxyl radical generation in cells submitted to the aforementioned treatments will be evaluated. The project has already been started and together with the research group of Prof. Dr. João H. G. Lago of the UFABC were identified, selected and purified natural compounds from the Brazilian flora that have potential characteristics to behave as inhibitors of typical 2-Cys Prx. The expression and purification conditions of the target proteins of the project have also been established and preliminary tests of inhibition of the peroxidase activity have led to the identification of a compound (C3) that presented a potent inhibitory capacity on yeast Tsa1 (45.5% ± 5.7%) and moderate toxicity on human Prx2 (10.3 ± 5.8%). Additional tests for confirmation of results by other methodologies are in progress.