Efeitos de compostos naturais sobre a atividade peroxidásica de AhpCs e sobrevivência de bactérias patogênicas
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2023-02-02
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Universidade Estadual Paulista (Unesp)
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A resistência de bactérias patogênicas a múltiplos antibióticos aumentou em todo o mundo nos últimos anos, exigindo a busca de novos compostos antibacterianos. Estudos indicam que ação convergente de algumas classes de antibióticos, independente do mecanismo de ação, é a geração de espécies reativas de oxigênio (EROS), o que contribui para a morte de patógenos bacterianos. As bactérias possuem várias enzimas antioxidantes capazes de decompor as EROs, entre elas, as peroxirredoxinas 2-Cys típicas, denominadas de AhpC em bactérias, são peroxidases altamente reativas e extremamente abundantes no ambiente celular, e estudos sugerem que a atividade dessa enzima seria um fator importante para virulência de determinadas cepas bacterianas. A alta reatividade de AhpC está relacionada a uma tríade catalítica, composta por dois resíduos polares (Thr/Ser e Arg) que mantêm o enxofre da cisteína catalítica, denominada de cisteína peroxidásica (CP) na forma tiolato (S-) e estão envolvidos no direcionamento e estabilização do substrato para permitir a substituição nucleofílica bimolecular química (SN2). Apesar da importância da AhpC em bactérias, até o presente momento nenhum inibidor foi identificado para estas enzimas. Por outro lado, alguns produtos naturais foram identificados como inibidores de isoformas humanas. De forma geral estes compostos apresentam como características comuns um esqueleto hidrofóbico volumoso e um sistema carbonílico capaz de realizar uma adição de tiól-Michael. Neste trabalho, avaliamos a atividade inibitória de compostos naturais oriundos da biota costeira do estado de São Paulo sobre a atividade peroxidase de AhpC de Pseudomonas aeruginosa (PaAhpC) e Staphylococcus epidermidis (SeAhpC), duas bactérias oportunistas Gram negativas e positivas, respectivamente, responsáveis por diversas infecções hospitalares. A seleção dos compostos foi efetuada com base em características funcionais e estruturais de compostos naturais identificados para isoformas humanas. Através de ensaios bioquímicos identificamos um ácido benzoico prenilado (CN-ABP1) oriundo de Piper crassinervium foi capaz de inibir a atividade peroxidásica de PaAhpC, mas não inibiu SeAhpC ou da isoforma humana (HsPrx2). Adicionalmente demonstramos que CN-ABP1 não é capaz de inibir outras tiól enzimas. A determinação do IC50 foi de 20.3 μM e por SDS PAGE mostramos que o composto não foi capaz de realizar uma adição de tiól-Michael, indicando um modo de inibição ainda não descrito para este grupo de proteínas. Resultados de simulações envolvendo docking molecular, indicam que o composto é estabilizado no sítio catalítico com aminoácidos da tríade catalítica através de ligações polares e um grande número de interações hidrofóbicas. Neste contexto os resultados obtidos nesta investigação levaram a identificação do primeiro composto natural oriundo da biota costeira brasileira com alta especificidade para AhpC bacteriana. Como consequências diretas da investigação iniciamos procedimentos com uma série de compostos similares a CN-ABP1 visando a identificação de características estruturais/funcionais envolvidas na inibição promovida por CN-ABP1. Ainda baseado na estrutura hidrofóbica alongada do composto hipotetizamos que este poderia mimetizar um substrato biológico como peróxidos derivados de ácidos graxos de cadeia longa e resultados obtidos revelaram que AhpC possui alta afinidade por este tipo de substrato com taxas de hiperoxidação de 1.06 ´ 106M-1s-1 indicando que estes possam ser inibidores biológicos de AhpC. Visando aprofundar o conhecimento da biologia estrutural da enzima realizamos ensaios de cristalização e obtivemos cristais passiveis de difração de raios-X. A determinação da estrutura destas proteínas deve auxiliar em um maior entendimento da interação entre CN-ABP1-PaAhpC abrindo a perspectiva da identificação de outros inibidores que guardem semelhança com CN-ABP1 ou hidroperóxidos derivados de lipídeos de cadeia longa, que podem em última instancia auxiliar no combate a bactérias patogênicas resistentes a múltiplos antibióticos.
The resistance of pathogenic bacteria to multiple antibiotics has increased worldwide in recent years, requiring the search for new antibacterial compounds. Recently, some studies have shown that distinct antibiotics with different targets have the convergent ability to produce reactive oxygen species (ROS), which contributes to the death of bacterial pathogens. Bacteria possess several antioxidant enzymes capable of breaking down ROS. Among them, the typical 2-Cys peroxiredoxins, called AhpC in bacteria, are highly reactive peroxidases that are extremely abundant in the cellular environment, and studies indicate that AhpC is associated with virulence. The high reactivity of AhpC is related to a catalytic triad, composed of two polar residues (Thr/Ser and Arg) that keep the sulfur of the catalytic cysteine, called cysteine peroxidase (CP) in the thiolate (S- ) form and are involved in the targeting and stabilization of the substrate to allow for chemical bimolecular nucleophilic substitution (SN2). Despite the importance of AhpC in bacteria, to date no inhibitors have been identified for these enzymes. On the other hand, some natural products have been identified as inhibitors of human isoforms. In general, these compounds present as common features a bulky hydrophobic skeleton and a carbonyl system capable of performing a thiol-Michael addition. In this work, we evaluated the inhibitory activity of natural compounds from the coastal biota of São Paulo state on the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC), two opportunistic Gram negative and positive bacteria, respectively, responsible for several hospital infections. The selection of compounds was made based on functional and structural characteristics of natural compounds identified for human isoforms. Through biochemical assays we identified a prenylated benzoic acid (CN-ABP1) from Piper crassinervium was able to inhibit the peroxidase activity of PaAhpC but did not inhibit SeAhpC or the human isoform (HsPrx2). Additionally we demonstrated that CN-ABP1 is not able to inhibit other thiol enzymes. The IC50 determination was 20.3 µM and by SDS PAGE we showed that the compound was not able to perform a thiol-Michael addition, indicating a mode of inhibition not yet described for this group of proteins. Computational simulations involving molecular docking, indicate that the compound is stabilized in the active site pocket by amino acids of the catalytic triad through polar and a large number of hydrophobic interactions. In this context, the results obtained in this investigation led to the identification of the first natural compound from the Brazilian coastal biota with high specificity for bacterial AhpC. As direct consequences of the investigation we initiated approaches with a series of compounds similar to CN-ABP1 aiming the identification of structural/functional characteristics involved in the inhibition promoted by CN-ABP1. Based on the elongated hydrophobic structure of the compound we hypothesized that it could mimics biological substrates such as long chain fatty acid hydroperoxides and initial results revealed that AhpC has high affinity for this type of substrate with hyperoxidation rates of 1.06 ´ 106 M-1 s-1 indicating that these compounds may act as biological inhibitors of AhpC. In order to deepen the knowledge of the structural biology of the enzyme we performed the enzyme crystallization assays and obtained crystals suitable to X-ray diffraction approaches. The determination of the structure of these proteins may help in a better understanding of the interaction between CN-ABP1- PaAhpC opening the perspective to identify other inhibitors that keep similarity with CN-ABP1 or long-chain hydroperoxides derivatives, which may, ultimately, assist the combat against pathogenic bacteria resistant to multiple antibiotics.
The resistance of pathogenic bacteria to multiple antibiotics has increased worldwide in recent years, requiring the search for new antibacterial compounds. Recently, some studies have shown that distinct antibiotics with different targets have the convergent ability to produce reactive oxygen species (ROS), which contributes to the death of bacterial pathogens. Bacteria possess several antioxidant enzymes capable of breaking down ROS. Among them, the typical 2-Cys peroxiredoxins, called AhpC in bacteria, are highly reactive peroxidases that are extremely abundant in the cellular environment, and studies indicate that AhpC is associated with virulence. The high reactivity of AhpC is related to a catalytic triad, composed of two polar residues (Thr/Ser and Arg) that keep the sulfur of the catalytic cysteine, called cysteine peroxidase (CP) in the thiolate (S- ) form and are involved in the targeting and stabilization of the substrate to allow for chemical bimolecular nucleophilic substitution (SN2). Despite the importance of AhpC in bacteria, to date no inhibitors have been identified for these enzymes. On the other hand, some natural products have been identified as inhibitors of human isoforms. In general, these compounds present as common features a bulky hydrophobic skeleton and a carbonyl system capable of performing a thiol-Michael addition. In this work, we evaluated the inhibitory activity of natural compounds from the coastal biota of São Paulo state on the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC), two opportunistic Gram negative and positive bacteria, respectively, responsible for several hospital infections. The selection of compounds was made based on functional and structural characteristics of natural compounds identified for human isoforms. Through biochemical assays we identified a prenylated benzoic acid (CN-ABP1) from Piper crassinervium was able to inhibit the peroxidase activity of PaAhpC but did not inhibit SeAhpC or the human isoform (HsPrx2). Additionally we demonstrated that CN-ABP1 is not able to inhibit other thiol enzymes. The IC50 determination was 20.3 µM and by SDS PAGE we showed that the compound was not able to perform a thiol-Michael addition, indicating a mode of inhibition not yet described for this group of proteins. Computational simulations involving molecular docking, indicate that the compound is stabilized in the active site pocket by amino acids of the catalytic triad through polar and a large number of hydrophobic interactions. In this context, the results obtained in this investigation led to the identification of the first natural compound from the Brazilian coastal biota with high specificity for bacterial AhpC. As direct consequences of the investigation we initiated approaches with a series of compounds similar to CN-ABP1 aiming the identification of structural/functional characteristics involved in the inhibition promoted by CN-ABP1. Based on the elongated hydrophobic structure of the compound we hypothesized that it could mimics biological substrates such as long chain fatty acid hydroperoxides and initial results revealed that AhpC has high affinity for this type of substrate with hyperoxidation rates of 1.06 ´ 106 M-1 s-1 indicating that these compounds may act as biological inhibitors of AhpC. In order to deepen the knowledge of the structural biology of the enzyme we performed the enzyme crystallization assays and obtained crystals suitable to X-ray diffraction approaches. The determination of the structure of these proteins may help in a better understanding of the interaction between CN-ABP1- PaAhpC opening the perspective to identify other inhibitors that keep similarity with CN-ABP1 or long-chain hydroperoxides derivatives, which may, ultimately, assist the combat against pathogenic bacteria resistant to multiple antibiotics.
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Peroxirredoxinas, Inibidores, Compostos naturais, Peróxidos orgânicos