Busca de inibidores do sistema peroxirredoxina 2-Cys típicas de eucariotos: avaliação funcional e estrutural
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Data
2021-08-20
Autores
Oliveira, Ana Laura Pires de
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Universidade Estadual Paulista (Unesp)
Resumo
Em infecções causadas por fungos patogênicos, o estresse oxidativo/nitrosativo promovido por células de defesa do hospedeiro e/ou pela ação de fungicidas, é utilizado para controlar a progressão do patógeno. Entretanto, em indivíduos imunocomprometidos, infecções invasivas e linhagens resistentes aos principais fungicidas, como por exemplo, a anfotericina B (AMB), representam um grave problema de saúde pública. A resistência destes patógenos está relacionada com difersos fatores, dentre eles, a presença de enzimas antioxidantes de alta eficiência, capazes de neutralizar os oxidantes produzidos pelo hospedeiro e pela administração de fungicidas. Neste contexto, um grupo de peroxidases denominadas Peroxirredoxinas 2-Cys típicas (Prx 2-Cys típicas) se destacam por decompor uma grande variedade de hidroperóxidos com alta especificidade e eficiência (k = 106 – 108 M-1s-1), estarem distribuídas nos mais diversos ambientes celulares e possuírem uma cisteína altamente reativa denominada cisteína peroxidásica (CP). Apesar das Prx 2-Cys típicas de fungos patogênicos serem um alvo bastante atraente para o combate de infecções fúngicas, nenhum inibidor de Prx 2-Cys típicas foi caracterizado para estes organismos. Por outro lado, já foram descritos inibidores destas enzimas para mamíferos, dentre eles, diterpenóides naturais como a Adenantina (Adn) e o JDA-202, que são capazes de inibir isoformas citosólicas das Prx 2-Cys típicas de humanos (Prx1 e Prx2), levando a remissão de tumores com baixa toxicidade para células sadias. É importante ressaltar que estudos que investigam inibidores de Prx 2-Cys típicas nunca atentaram para uma avaliação comparativa com enzimas de outros organismos. Nosso grupo de pesquisa demonstrou que a Adn é capaz de inibir as isoformas citosólicas Tsa1 e Tsa2 de Saccharomyces cerevisiae, um organismo modelo para estudos bioquímicos e genéticos. Investigações realizadas no âmbito deste trabalho revelaram que Tsa1 apresenta alta identidade com isoformas citosólicas de leveduras patogênicas dos gêneros Candida e Cryptococcus, revelando que esta levedura é um bom modelo para estudo destes micro-organismos. Os objetivos deste trabalho residiram na busca por compostos naturais da biota costeira de São Paulo com atividade inibitória sobre Prx 2-Cys típicas de fungos, utilizando como modelo Tsa1 de S. cerevisiae de forma comparativa com Prx2, a isoforma mais abundante de Prx 2-Cys típicas de humano e na utilização desta levedura para ensaios in vivo do potencial fungicida dos compostos identificados como inibidores de Tsa1, assim como inibidores + fungicidas amplamente utilizados em tratamentos clínicos (ex: AMB). Neste contexo, foram avaliadas as propriedades inibitórias da Adn, assim como de terpenóides ent-caurânos e lactonas sesquiterpenicas da biota brasileira. Nossos resultados revelaram que a Adn, como esperado, é capaz de inibir eficientemente a Prx2 humana e redeterminamos seu IC50 como 2 ± 0.13 μM. Em relação aos compostos naturais, nossos resultados revelaram que os compostos NC-EK2, NC-LS1, NC-LS5, NC-HP1 e NC-ABP1 foram capazes de exercer atividade inibitória sobre as Prx 2-Cys típicas por mecanismos aparentemente distintos. Também demonstramos que NC-LS5 é capaz de execer atividade inibitória sobre o crescimento de células de levedura. Evidências obtidas por SDS-PAGE indicam que NC-LS1 é capaz de impedir a formação de dissulfetos, o que pode ser atribuído aos sistemas carbonilicos ,-insaturados presentes em sua estrutura, capazes de efetuar uma adição de Michael com tióis das enzimas, indicando uma inibição irreversível a exemplo de Adn. Por outro lado, o composto NC-EK2 não possui estes sistemas sugerindo uma inibição do tipo competitiva, entretanto, análises adicionais são necessárias para se chegar a qualquer observação mais conclusiva. Foram realizados procedimentos de docking molecular sobre as estruturas de Tsa1 e Prx2, visando um melhor entendimento das interações moleculares entre os compostos identificados como inibidores. Inicialmente, efetuamos as simulações utilizando Tsa1 e os inibidores Adn e NC-LS1, onde Adn foi utilizada por já existirem dados comparativos de docking utilizando Prx1 de humanos e ambos são capazes de bloquear as cisteínas. Desta forma, para ambos os compostos, se esperaria resultados positivos em regiões próximas as cisteínas. Os resultados obtidos indicam que o posicionamento dos compostos ocorreu integralmente na região do sitio ativo próximo à CP e que as interações para estabilização dos ligantes são majoritarimante de caráter hidrofóbico envolvendo átomos das cadeias laterais dos resíduos Leu41, Phe45, Leu142, Val167, Pro181 e Tyr77 e também das cadeias principais de Phe45, Leu142. A maioria dos resíduos identificados em Tsa1 são equivalentes aos resíduos já relatados na literatura, identificados por docking em Prx1 de humanos, assim como aos resíduos que interagem com um ligante cristalizado juntamente com Tsa1 pelo nosso grupo de pesquisa (PDB: 3SBC), o que gera uma maior confiabilidade dos dados obtidos. De forma geral, os resultados obtidos neste projeto representam a primeira abordagem comparativa da atividade inibitória de compostos naturais sobre 2-Cys Prx típicas humana e de fungo incluindo a identificação de novos compostos da biota costeira brasileira.
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.
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.
Descrição
Palavras-chave
Peroxirredoxinas, Leveduras, Biologia molecular, Inibidores, Humanos