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: 2018-01-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / 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. / Os dados epidemiol?gicos relacionados ? tuberculose (TB) indicam que ainda existe uma carga elevada desta doen?a no mundo todo. Al?m disso, o surgimento de cepas resistentes aos f?rmacos imp?e uma nova amea?a na preven??o da propaga??o da TB. Portanto, ? fundamental buscar continuamente novos candidatos para o desenvolvimento de medicamentos. No Cap?tulo 2 desta tese ? apresentado o composto IQG-607, que ? um complexo met?lico que tem sido reportado como uma mol?cula anti-TB promissora contra cepas de M. tuberculosis resistentes ? isoniazida (INH). Estudos pr?vios sugeriram que o composto inibe a enzima selvagem trans-2-enoil-[ACP] redutase dependente de NADH (InhA) e algumas das suas mutantes estruturais, na aus?ncia de NAD+ ou NADH e sem necessitar da enzima KatG. O IQG-607 tamb?m mostrou um perfil toxicol?gico favor?vel in vivo, com uma menor toxicidade em compara??o ? INH. No entanto, ainda existe uma lacuna em rela??o ? atividade do IQG-607 contra cepas que carregam muta??es no gene katG, as quais s?o as altera??es gen?ticas mais comuns em isolados cl?nicos resistentes ? INH. Sendo assim, este estudo focou em elucidar o mecanismo de resist?ncia (MOR) do Mycobacterium tuberculosis, o principal agente causador da TB, ao composto IQG-607. Primeiramente, a concentra??o inibit?ria m?nima (MIC) do IQG-607 foi estabelecida contra oito isolados cl?nicos multirresistentes a f?rmacos (MDR), os quais foram resistentes ao nosso composto. Ent?o, mutantes espont?neos foram selecionados, usando-se altas concentra??es do composto em meio ?gar 7H10, e seus genomas completos foram sequenciados; os resultados revelaram altera??es no gene katG. Uma cepa laboratorial, carregando o gene katG(S315T) mutante, foi desenvolvida para acessar o efeito desta ?nica muta??o na atividade do composto, atrav?s da determina??o de MIC e por meio de um modelo de infec??o de macr?fagos. Os resultados mostraram que essa muta??o de fato foi suficiente para conferir resist?ncia ao IQG-607. Finalmente, a resist?ncia observada para a cepa que expressa a prote?na InhA(S94A) mutante sugeriu que o IQG-607 tem esta enzima como seu alvo molecular. No Cap?tulo 3, dois novos compostos, denominados Labio-16 e Labio-17, s?o apresentados, os quais foram previamente selecionados para interagir e inibir a enzima InhA, e que j? tinham mostrado ser ativos contra a cepa H37Rv de M. tuberculosis. Um conjunto de experimentos foi conduzido para elucidar os seus mecanismos de a??o (MOA) e para compreender o MOR do M. tuberculosis contra eles, similar ?quele usado para estudar o IQG-607. At? o momento, os resultados sugerem que a InhA n?o ? o alvo molecular desses compostos. Outros experimentos est?o em andamento em nosso laborat?rio, para avaliar em um modelo murino da infec??o da TB os seus potenciais como candidatos a f?rmacos anti-TB.