Search for inhibitors of the eukaryotic typical 2-Cys peroxiredoxin system: functional and structural evaluation

Author(s)

Ana Laura Pires de Oliveira

• Document type: Master's Dissertation
• Press: São Vicente. 2021-09-16.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. São Vicente
• Defense date: 2021-08-20
• Advisor: Marcos Antonio de Oliveira

Abstract

In infections caused by pathogenic fungi, oxidative/nitrosative stress promoted by host defense cells and/or by the action of fungicides is used to control pathogen progression. However, in immunocompromised individuals, invasive infections and strains resistant to the main fungicides, such as amphotericin B (AMB), represent a serious public health problem. The resistance of these pathogens is related to several factors, among them the presence of antioxidant enzymes of high efficiency, capable of neutralizing the oxidants produced by the host and by the administration of fungicides. In this context, a group of peroxidases called Typical 2-Cys Peroxiredoxins (Typical Prx 2-Cys) stand out for breaking down a wide variety of hydroperoxides with high specificity and efficiency (k = 106 – 108 M-1s-1), being distributed in the most diverse cellular environments and possessing a highly reactive cysteine called peroxidase cysteine (CP). Although the typical Prx 2-Cys of pathogenic fungi is a very attractive target for fighting fungal infections, no inhibitors of typical Prx 2-Cys have 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 able to inhibit cytosolic isoforms of the typical human Prx 2-Cys (Prx1 and Prx2), leading to tumor remission with low toxicity to healthy cells. Importantly, studies investigating inhibitors of typical Prx 2-Cys have never paid attention to a comparative evaluation with enzymes from other organisms. Our research group has demonstrated that Adn is able to inhibit the cytosolic isoforms Tsa1 and Tsa2 of Saccharomyces cerevisiae, a model organism for biochemical and genetic studies. Investigations performed as part of this work revealed that Tsa1 shows high identity with cytosolic isoforms of pathogenic yeasts of the genera Candida and Cryptococcus, revealing that this yeast is a good model for studying these microorganisms. The objectives of this work were in the search for natural compounds from the coastal biota of São Paulo with inhibitory activity on Prx 2-Cys typical of fungi, using as model Tsa1 of S. cerevisiae in a comparative way with Prx2, the most abundant isoform of Prx 2-Cys typical of human and in the use of this yeast for in vivo assays of the fungicidal potential of the compounds identified as Tsa1 inhibitors, as well as inhibitors + fungicides widely used in clinical treatments (e.g. AMB). In this context, the inhibitory properties of Adn, as well as ent-cauranic terpenoids and sesquiterpene lactones from Brazilian biota, were evaluated. Our results revealed that Adn, as expected, is able to efficiently inhibit human Prx2 and we redetermined its IC50 as 2 ± 0.13 μM. Regarding natural compounds, our results revealed that compounds NC-EK2, NC-LS1, NC-LS5, NC-HP1 and NC-ABP1 were able to exert inhibitory activity on typical Prx 2-Cys by apparently distinct mechanisms. We also demonstrated that NC-LS5 is able to exert inhibitory activity on yeast cell growth. Evidence obtained by SDS-PAGE indicates that NC-LS1 is able to prevent the formation of disulfides, which can be attributed to the carbonyl α-β-unsaturated systems present in its structure, able to effect a Michael addition with thiols of the enzymes, indicating an irreversible inhibition like Adn. On the other hand, the NCEK2 compound does not have these systems suggesting a competitive type inhibition, however, further analysis is needed to arrive at any more conclusive observation. Molecular docking procedures were performed on the structures of Tsa1 and Prx2, aiming at a better understanding of the molecular interactions between the compounds identified as inhibitors. Initially, we performed the simulations using Tsa1 and the inhibitors Adn and NC-LS1, where Adn was used because there are already comparative docking data using Prx1 from humans and both are able to block cysteines. Thus, for both compounds, one would expect positive results in regions near the cysteines. The results obtained indicate that the positioning of the compounds occurred entirely in the region of the active site near the CP and that the interactions for stabilization of the ligands are mostly of hydrophobic character involving atoms of the side chains of residues Leu41, Phe45, Leu142, Val167, Pro181 and Tyr77 and also of the main chains of Phe45, Leu142. Most of the residues identified in Tsa1 are equivalent to residues already reported in the literature, identified by docking in human Prx1, as well as to residues interacting with a ligand crystallized together with Tsa1 by our research group (PDB: 3SBC), which generates a higher reliability of the obtained data. Overall, the results obtained in this project represent the first comparative approach to the inhibitory activity of natural compounds on human and fungal 2-Cys Prx including the identification of new compounds from the Brazilian coastal biota. (AU)

• FAPESP's process: 19/04054-4 - Search of inhibitors of typical 2-Cys peroxirredoxin system from eukaryotes: functional and structural evaluation
• Grantee: Ana Laura Pires de Oliveira
• Support Opportunities: Scholarships in Brazil - Master