Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana
Autor(a) principal: | |
---|---|
Data de Publicação: | 2018 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da PUC_RS |
Texto Completo: | http://tede2.pucrs.br/tede2/handle/tede/7871 |
Resumo: | Epidemiologic data regarding tuberculosis (TB) show that there is still a high burden of this disease worldwide. In addition, the emergence of drug-resistant strains imposes a new threat in preventing TB spread. Therefore, it is pivotal to continuously find new candidates for drug development. In the Chapter 2 of this thesis, the compound IQG-607 is presented, which is a metal complex that has been reported as a promising anti-TB molecule against isoniazid (INH)-resistant strains of M. tuberculosis. Previous studies suggested that the compound inhibits both the wild-type NADH-dependent trans-2-enoyl-[ACP] reductase (InhA) enzyme and some of its structural mutants in the absence of NAD+ or NADH and without requiring KatG enzyme. IQG-607 has also shown a favorable toxicological profile in vivo, with a considerable lesser toxicity compared to INH. However, there is still a gap regarding the activity of IQG-607 against strains carrying mutations in the katG gene, which are the most common genetic alterations in clinical isolates resistant to INH. Therefore, this study focused in elucidating the mechanism of resistance (MOR) of the Mycobacterium tuberculosis, the main causative agent of TB, to compound IQG-607. First the minimum inhibitory concentration (MIC) of IQG-607 was established against eight multi-drug resistant (MDR) clinical isolates, which were resistant to our compound. Then spontaneous mutants were selected using high concentrations of compound in 7H10 agar medium, and their whole genomes were sequenced; the results revealed alterations in the katG gene. A laboratory strain carrying the mutant katG(S315T) gene was developed to assess the effect of this single mutation in the compound activity both by MIC determination and by a macrophage infection model. Results showed that this mutation was indeed sufficient to confer resistance to IQG-607. Finally, the resistance observed for a strain expressing a mutant InhA(S94A) protein suggested that IQG-607 has this enzyme as its molecular target. In the Chapter 3, two new compounds, called Labio-16 and Labio-17, are presented, which were previously selected to interact and inhibit the InhA enzyme and that had already shown to be active against M. tuberculosis H37Rv strain. A set of experiments were conducted to elucidate their mechanism of action (MOA) and to understand the MOR of the M. tuberculosis against them, similar to those carried for studying IQG-607. So far, results suggested that the InhA is not the molecular target of these compounds. Other experiments are undergoing in our laboratory to evaluate in a murine model of TB infection their potential as anti-TB drug candidates. |
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Bizarro, Cristiano Valimhttp://lattes.cnpq.br/8237569228020224Villela, Anne Drumondhttp://lattes.cnpq.br/1888884411320073http://lattes.cnpq.br/7502461555142614Abbadi, Bruno Lopes2018-03-06T16:21:42Z2018-01-19http://tede2.pucrs.br/tede2/handle/tede/7871Epidemiologic data regarding tuberculosis (TB) show that there is still a high burden of this disease worldwide. In addition, the emergence of drug-resistant strains imposes a new threat in preventing TB spread. Therefore, it is pivotal to continuously find new candidates for drug development. In the Chapter 2 of this thesis, the compound IQG-607 is presented, which is a metal complex that has been reported as a promising anti-TB molecule against isoniazid (INH)-resistant strains of M. tuberculosis. Previous studies suggested that the compound inhibits both the wild-type NADH-dependent trans-2-enoyl-[ACP] reductase (InhA) enzyme and some of its structural mutants in the absence of NAD+ or NADH and without requiring KatG enzyme. IQG-607 has also shown a favorable toxicological profile in vivo, with a considerable lesser toxicity compared to INH. However, there is still a gap regarding the activity of IQG-607 against strains carrying mutations in the katG gene, which are the most common genetic alterations in clinical isolates resistant to INH. Therefore, this study focused in elucidating the mechanism of resistance (MOR) of the Mycobacterium tuberculosis, the main causative agent of TB, to compound IQG-607. First the minimum inhibitory concentration (MIC) of IQG-607 was established against eight multi-drug resistant (MDR) clinical isolates, which were resistant to our compound. Then spontaneous mutants were selected using high concentrations of compound in 7H10 agar medium, and their whole genomes were sequenced; the results revealed alterations in the katG gene. A laboratory strain carrying the mutant katG(S315T) gene was developed to assess the effect of this single mutation in the compound activity both by MIC determination and by a macrophage infection model. Results showed that this mutation was indeed sufficient to confer resistance to IQG-607. Finally, the resistance observed for a strain expressing a mutant InhA(S94A) protein suggested that IQG-607 has this enzyme as its molecular target. In the Chapter 3, two new compounds, called Labio-16 and Labio-17, are presented, which were previously selected to interact and inhibit the InhA enzyme and that had already shown to be active against M. tuberculosis H37Rv strain. A set of experiments were conducted to elucidate their mechanism of action (MOA) and to understand the MOR of the M. tuberculosis against them, similar to those carried for studying IQG-607. So far, results suggested that the InhA is not the molecular target of these compounds. Other experiments are undergoing in our laboratory to evaluate in a murine model of TB infection their potential as anti-TB drug candidates.Os dados epidemiológicos relacionados à tuberculose (TB) indicam que ainda existe uma carga elevada desta doença no mundo todo. Além disso, o surgimento de cepas resistentes aos fármacos impõe uma nova ameaça na prevenção da propagação da TB. Portanto, é fundamental buscar continuamente novos candidatos para o desenvolvimento de medicamentos. No Capítulo 2 desta tese é apresentado o composto IQG-607, que é um complexo metálico que tem sido reportado como uma molécula anti-TB promissora contra cepas de M. tuberculosis resistentes à isoniazida (INH). Estudos prévios sugeriram que o composto inibe a enzima selvagem trans-2-enoil-[ACP] redutase dependente de NADH (InhA) e algumas das suas mutantes estruturais, na ausência de NAD+ ou NADH e sem necessitar da enzima KatG. O IQG-607 também mostrou um perfil toxicológico favorável in vivo, com uma menor toxicidade em comparação à INH. No entanto, ainda existe uma lacuna em relação à atividade do IQG-607 contra cepas que carregam mutações no gene katG, as quais são as alterações genéticas mais comuns em isolados clínicos resistentes à INH. Sendo assim, este estudo focou em elucidar o mecanismo de resistência (MOR) do Mycobacterium tuberculosis, o principal agente causador da TB, ao composto IQG-607. Primeiramente, a concentração inibitória mínima (MIC) do IQG-607 foi estabelecida contra oito isolados clínicos multirresistentes a fármacos (MDR), os quais foram resistentes ao nosso composto. Então, mutantes espontâneos foram selecionados, usando-se altas concentrações do composto em meio ágar 7H10, e seus genomas completos foram sequenciados; os resultados revelaram alterações no gene katG. Uma cepa laboratorial, carregando o gene katG(S315T) mutante, foi desenvolvida para acessar o efeito desta única mutação na atividade do composto, através da determinação de MIC e por meio de um modelo de infecção de macrófagos. Os resultados mostraram que essa mutação de fato foi suficiente para conferir resistência ao IQG-607. Finalmente, a resistência observada para a cepa que expressa a proteína InhA(S94A) mutante sugeriu que o IQG-607 tem esta enzima como seu alvo molecular. No Capítulo 3, dois novos compostos, denominados Labio-16 e Labio-17, são apresentados, os quais foram previamente selecionados para interagir e inibir a enzima InhA, e que já tinham mostrado ser ativos contra a cepa H37Rv de M. tuberculosis. Um conjunto de experimentos foi conduzido para elucidar os seus mecanismos de ação (MOA) e para compreender o MOR do M. tuberculosis contra eles, similar àquele usado para estudar o IQG-607. Até o momento, os resultados sugerem que a InhA não é o alvo molecular desses compostos. Outros experimentos estão em andamento em nosso laboratório, para avaliar em um modelo murino da infecção da TB os seus potenciais como candidatos a fármacos anti-TB.Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-05T13:21:28Z No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5)Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-03-06T16:15:19Z (GMT) No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5)Made available in DSpace on 2018-03-06T16:21:42Z (GMT). No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5) Previous issue date: 2018-01-19Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfhttp://tede2.pucrs.br:80/tede2/retrieve/171005/TES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.jpghttp://tede2.pucrs.br:80/tede2/retrieve/174247/TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.jpgporPontifícia Universidade Católica do Rio Grande do SulPrograma de Pós-Graduação em Biologia Celular e MolecularPUCRSBrasilEscola de CiênciasResistência a AntibióticoDesenvolvimento de FármacosMICMutantes EspontâneosTuberculoseSequenciamento Completo do GenomaCIENCIAS BIOLOGICAS::BIOLOGIA GERALElucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacterianainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisTrabalho será publicado como artigo ou livro12 meses06/03/20198198246930096637360500500600-16345593859312446972075167498588264571info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da PUC_RSinstname:Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)instacron:PUC_RSORIGINALTES_BRUNO_LOPES_ABBADI_COMPLETO.pdfTES_BRUNO_LOPES_ABBADI_COMPLETO.pdfapplication/pdf4934736http://tede2.pucrs.br/tede2/bitstream/tede/7871/5/TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf58e4c3c86d9fb96bb1476ae3c18bfdf0MD55THUMBNAILTES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.jpgTES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.jpgimage/jpeg4081http://tede2.pucrs.br/tede2/bitstream/tede/7871/4/TES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.jpg6717485a878e2bf8828a9173ae27ae6eMD54TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.jpgTES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.jpgimage/jpeg5618http://tede2.pucrs.br/tede2/bitstream/tede/7871/7/TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.jpge9ceb689a2eafb01be7d2a576928d94bMD57TEXTTES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.txtTES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.txttext/plain1675http://tede2.pucrs.br/tede2/bitstream/tede/7871/3/TES_BRUNO_LOPES_ABBADI_CONFIDENCIAL.pdf.txte4b0b4e03509ab7f9133eff726018d12MD53TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.txtTES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.txttext/plain146901http://tede2.pucrs.br/tede2/bitstream/tede/7871/6/TES_BRUNO_LOPES_ABBADI_COMPLETO.pdf.txt2baf2ae8d36aeb7064bce65843664acbMD56LICENSElicense.txtlicense.txttext/plain; charset=utf-8610http://tede2.pucrs.br/tede2/bitstream/tede/7871/1/license.txt5a9d6006225b368ef605ba16b4f6d1beMD51tede/78712019-03-12 20:01:45.908oai:tede2.pucrs.br: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Biblioteca Digital de Teses e Dissertaçõeshttp://tede2.pucrs.br/tede2/PRIhttps://tede2.pucrs.br/oai/requestbiblioteca.central@pucrs.br||opendoar:2019-03-12T23:01:45Biblioteca Digital de Teses e Dissertações da PUC_RS - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)false |
dc.title.por.fl_str_mv |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
title |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
spellingShingle |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana Abbadi, Bruno Lopes Resistência a Antibiótico Desenvolvimento de Fármacos MIC Mutantes Espontâneos Tuberculose Sequenciamento Completo do Genoma CIENCIAS BIOLOGICAS::BIOLOGIA GERAL |
title_short |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
title_full |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
title_fullStr |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
title_full_unstemmed |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
title_sort |
Elucidação do mecanismo de resistência de Mycobacterium tuberculosis frente a novos compostos com atividade antimicobacteriana |
author |
Abbadi, Bruno Lopes |
author_facet |
Abbadi, Bruno Lopes |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Bizarro, Cristiano Valim |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/8237569228020224 |
dc.contributor.advisor-co1.fl_str_mv |
Villela, Anne Drumond |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/1888884411320073 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/7502461555142614 |
dc.contributor.author.fl_str_mv |
Abbadi, Bruno Lopes |
contributor_str_mv |
Bizarro, Cristiano Valim Villela, Anne Drumond |
dc.subject.por.fl_str_mv |
Resistência a Antibiótico Desenvolvimento de Fármacos MIC Mutantes Espontâneos Tuberculose Sequenciamento Completo do Genoma |
topic |
Resistência a Antibiótico Desenvolvimento de Fármacos MIC Mutantes Espontâneos Tuberculose Sequenciamento Completo do Genoma CIENCIAS BIOLOGICAS::BIOLOGIA GERAL |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOLOGIA GERAL |
description |
Epidemiologic data regarding tuberculosis (TB) show that there is still a high burden of this disease worldwide. In addition, the emergence of drug-resistant strains imposes a new threat in preventing TB spread. Therefore, it is pivotal to continuously find new candidates for drug development. In the Chapter 2 of this thesis, the compound IQG-607 is presented, which is a metal complex that has been reported as a promising anti-TB molecule against isoniazid (INH)-resistant strains of M. tuberculosis. Previous studies suggested that the compound inhibits both the wild-type NADH-dependent trans-2-enoyl-[ACP] reductase (InhA) enzyme and some of its structural mutants in the absence of NAD+ or NADH and without requiring KatG enzyme. IQG-607 has also shown a favorable toxicological profile in vivo, with a considerable lesser toxicity compared to INH. However, there is still a gap regarding the activity of IQG-607 against strains carrying mutations in the katG gene, which are the most common genetic alterations in clinical isolates resistant to INH. Therefore, this study focused in elucidating the mechanism of resistance (MOR) of the Mycobacterium tuberculosis, the main causative agent of TB, to compound IQG-607. First the minimum inhibitory concentration (MIC) of IQG-607 was established against eight multi-drug resistant (MDR) clinical isolates, which were resistant to our compound. Then spontaneous mutants were selected using high concentrations of compound in 7H10 agar medium, and their whole genomes were sequenced; the results revealed alterations in the katG gene. A laboratory strain carrying the mutant katG(S315T) gene was developed to assess the effect of this single mutation in the compound activity both by MIC determination and by a macrophage infection model. Results showed that this mutation was indeed sufficient to confer resistance to IQG-607. Finally, the resistance observed for a strain expressing a mutant InhA(S94A) protein suggested that IQG-607 has this enzyme as its molecular target. In the Chapter 3, two new compounds, called Labio-16 and Labio-17, are presented, which were previously selected to interact and inhibit the InhA enzyme and that had already shown to be active against M. tuberculosis H37Rv strain. A set of experiments were conducted to elucidate their mechanism of action (MOA) and to understand the MOR of the M. tuberculosis against them, similar to those carried for studying IQG-607. So far, results suggested that the InhA is not the molecular target of these compounds. Other experiments are undergoing in our laboratory to evaluate in a murine model of TB infection their potential as anti-TB drug candidates. |
publishDate |
2018 |
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2018-03-06T16:21:42Z |
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2018-01-19 |
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