Planejamento in silico e síntese de peptídeos derivados da proteína YjhX: estudos de inibição da atividade de DNA topoisomerases
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Data
2020-02-19
Autores
Brito, Lorrane Davi
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Universidade Estadual Paulista (Unesp)
Resumo
O recém-descoberto sistema YjhX-YjhQ bacteriano é um sistema toxina-antitoxina (TA) do tipo II (formado por duas proteínas) que apresenta um grande potencial a ser estudado com o intuito de desenvolver potenciais fármacos, tanto com ação antimicrobiana como antitumoral. A toxina YjhX é uma pequena proteína de 85 resíduos de aminoácidos, sem homologia com outras toxinas e descrita como a primeira proteína endógena, de efeito neutralizante, específica para topoisomerase IA. A antitoxina YjhQ (181 resíduos de aminoácidos) é capaz de neutralizar a citotoxicidade de YjhX, pela formação de um complexo estável com a toxina. YjhX possui atividade citotóxica na replicação do DNA e na síntese do RNA, promovendo assim a interrupção do crescimento e consequente morte celular de E. coli. Algumas evidências tem sugerido que a interação toxina-enzima ocorre na extremidade Nterminal da toxina. Embora esta toxina possa ser encontrada em um grande número de microrganismos, ainda apresenta mecanismos de citotoxicidade e funções celulares a serem elucidadas. Neste contexto, este trabalho teve como objetivo planejamento in silico e a síntese de peptídeos derivados de YjhX e avaliar a capacidade inibitória de diferentes topoisomerases. Com base nas informações estruturais, obtidas por modelagem e dinâmica molecular, quatro sequências peptídicas derivadas de YjhX foram projetadas e sintetizadas pela metodologia da fase sólida. As sequências foram analisadas e purificadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. A capacidade de inibição destes peptídeos sobre a atividade de diferentes topoisomerases foi avaliada por ensaios de eletroforese em gel. Ensaios de inibição do crescimento bacteriano, hemólise e viabilidade celular também foram realizados. Os peptídeos testados, não apresentaram atividade inibitória frente a DNA topoisomerases tipo II, mas inibiram a atividade da topoisomerases tipo I bacteriana. YjhXw-14I foi o único capaz de inibir 100% a atividade topo IA de M. tuberculosis na concentração de 100 μmol.L-1. Os ensaios microbiológicos envolvendo Klebsiella pneumoniae e Streptococcus agalactiae mostraram baixa atividade antimicrobiana. Embora determinadas sequências tenham apresentado um índice de inibição superior a 50%, os resultados não são satisfatórios por estarem abaixo dos 90% de inibição. Os peptídeos estudados não são tóxicos para células humanas, tendo em vista os resultados nos ensaios de hemólise e viabilidade celular. A partir dos resultados obtidos nesse trabalho, conclui-se que a α-hélice N-terminal de YjhX não deve participar do processo de interação com a enzima e consequentemente do processo inibitório, ou que o domínio 1 da topo IA não é aquele mais provável para a interação com a toxina.
The newly discovered bacterial YjhX-YjhQ system is a type II toxin-antitoxin (TA) system (formed by two proteins) that has great potential to be studied in order to develop drug products, both with antimicrobial and anti-tumor action. The YjhX toxin is a small protein of 85 amino acid residues, without homology with other toxins and used as the first endogenous protein, with a neutralizing effect, specific for topoisomerase IA. The YjhQ antitoxin (181 amino acid residues) is able to neutralize the cytotoxicity of YjhX, by forming a stable complex with the toxin. YjhX has cytotoxic activity on DNA replication and RNA synthesis, thus promoting an interruption of growth and consequent cell death of E. coli. Some evidences suggested that the enzyme-toxin interaction occur at the N-terminal end of the toxin. Although this toxin can be found in a large number of microorganisms, there are still mechanisms of cytotoxicity and cellular functions to be elucidated. In this context, this work aimed at in silico planning and the detection of peptides derived from YjhX and evaluation of the inhibitory capacity of different topoisomerases. Based on the structural information, obtained by molecular modeling and molecular dynamics, four peptides sequences, derived from YjhX, were proposed and synthesized by solid phase peptide synthesis. The sequences were analyzed and purified by high performance liquid chromatography and characterized by mass spectrometry. The ability of these peptides to inhibit the activity of different topoisomerases was evaluated by gel electrophoresis assays. Evaluation of bacterial development inhibition, hemolysis, viability was also performed. The proposed peptides did not show inhibitory activity against DNA topoisomerases type II, but they inhibited the activity of bacterial type I topoisomerases. YjhXw-14I was the only one capable of inhibiting 100% of the top IA activity of M. tuberculosis at a concentration of 100 μmol.L- 1. Microbiological tests involving Klebsiella pneumoniae and Streptococcus agalactiae showed low antimicrobial activity. Although the sequences showed an inhibition index greater than 50%, the results are not satisfactory because they are below 90% inhibition. The studied peptides are not toxic to human cells, considering the results caused by hemolysis and cell viability tests. From the results obtained in this work, it is concluded that the N-helical terminal of YjhX should not participate in the interaction process with enzymes and, consequently, in the inhibitory process, or that domain 1 of the top of the IA is not the most likely to interact with toxin.
The newly discovered bacterial YjhX-YjhQ system is a type II toxin-antitoxin (TA) system (formed by two proteins) that has great potential to be studied in order to develop drug products, both with antimicrobial and anti-tumor action. The YjhX toxin is a small protein of 85 amino acid residues, without homology with other toxins and used as the first endogenous protein, with a neutralizing effect, specific for topoisomerase IA. The YjhQ antitoxin (181 amino acid residues) is able to neutralize the cytotoxicity of YjhX, by forming a stable complex with the toxin. YjhX has cytotoxic activity on DNA replication and RNA synthesis, thus promoting an interruption of growth and consequent cell death of E. coli. Some evidences suggested that the enzyme-toxin interaction occur at the N-terminal end of the toxin. Although this toxin can be found in a large number of microorganisms, there are still mechanisms of cytotoxicity and cellular functions to be elucidated. In this context, this work aimed at in silico planning and the detection of peptides derived from YjhX and evaluation of the inhibitory capacity of different topoisomerases. Based on the structural information, obtained by molecular modeling and molecular dynamics, four peptides sequences, derived from YjhX, were proposed and synthesized by solid phase peptide synthesis. The sequences were analyzed and purified by high performance liquid chromatography and characterized by mass spectrometry. The ability of these peptides to inhibit the activity of different topoisomerases was evaluated by gel electrophoresis assays. Evaluation of bacterial development inhibition, hemolysis, viability was also performed. The proposed peptides did not show inhibitory activity against DNA topoisomerases type II, but they inhibited the activity of bacterial type I topoisomerases. YjhXw-14I was the only one capable of inhibiting 100% of the top IA activity of M. tuberculosis at a concentration of 100 μmol.L- 1. Microbiological tests involving Klebsiella pneumoniae and Streptococcus agalactiae showed low antimicrobial activity. Although the sequences showed an inhibition index greater than 50%, the results are not satisfactory because they are below 90% inhibition. The studied peptides are not toxic to human cells, considering the results caused by hemolysis and cell viability tests. From the results obtained in this work, it is concluded that the N-helical terminal of YjhX should not participate in the interaction process with enzymes and, consequently, in the inhibitory process, or that domain 1 of the top of the IA is not the most likely to interact with toxin.
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Palavras-chave
YjhQ antitoxin, YjhX toxin, Antitoxina YjhQ, Peptídeos, Topoisomerase, Toxina YjhX, Peptides