Detalhes bibliográficos
Ano de defesa: |
2020 |
Autor(a) principal: |
Barsé, Laísa Quadros
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Orientador(a): |
Bizarro, Cristiano Valim
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Banca de defesa: |
Não Informado pela instituição |
Tipo de documento: |
Dissertação
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Tipo de acesso: |
Acesso aberto |
Idioma: |
por |
Instituição de defesa: |
Pontifícia Universidade Católica do Rio Grande do Sul
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Programa de Pós-Graduação: |
Programa de Pós-Graduação em Biologia Celular e Molecular
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Departamento: |
Escola de Ciências
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País: |
Brasil
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Palavras-chave em Português: |
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Área do conhecimento CNPq: |
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Link de acesso: |
http://tede2.pucrs.br/tede2/handle/tede/9084
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Resumo: |
Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis (Mtb) and is responsible for millions of deaths. Moreover, new Mtb strains resistant to TB drugs are emerging and spreading. In particular, the main first-line TB drug, isoniazid (INH), must be activated inside mycobacterial cells by the catalase-peroxidase enzyme KatG to exert its antimicrobial activity, and mutations on the katG gene are a major cause of INH resistance in clinics. The metal-containing compound IQG-607 is an INH analogue that was developed to inhibit the same target of INH, the FASII enzyme enoyl-ACP-reductase (InhA), without requiring activation by the KatG. However, we showed recently that inside mycobacterial cells the activity of IQG-607 is also dependent on KatG. Hence, this compound might also be activated by KatG, requiring its activation to inhibit InhA. Therefore, we evaluated the ability of recombinant MtKatG to use IQG-607 as a substrate in oxidation reactions and for adduct formation with NAD+. A recombinant MtKatG was produced in E. coli and purified in a 3-step protocol to obtain a homogenous protein. An HPLC method was optimized to monitor both oxidation and adduct products, and our assay system was validated by performing control reactions using INH as a substrate. We found that the metal-based compound IQG-607 is not a substrate for recombinant MtKatG under all tested conditions. Based on our results, we suggest that IQG-607 might behave as a pre-prodrug, releasing the INH moiety inside mycobacterial cells, which then requires KatG activation to form the active INH-NAD adduct. |