Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-23T14:56:57Z No. of bitstreams: 1 DIS_FERNANDA_TEIXEIRA_SUBTIL_COMPLETO.pdf: 1495988 bytes, checksum: 70f1cb1dccabfa7c302effb51db76e03 (MD5) / Made available in DSpace on 2017-06-23T14:56:57Z (GMT). No. of bitstreams: 1 DIS_FERNANDA_TEIXEIRA_SUBTIL_COMPLETO.pdf: 1495988 bytes, checksum: 70f1cb1dccabfa7c302effb51db76e03 (MD5) Previous issue date: 2017-03-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The high incidence of tuberculosis is a great concern worldwide. Different strategies are being developed by the World Health Organization to fight tuberculosis. Amongst the three pillars that are part of the End TB Strategy, we can highlight the intensive research and innovation pillar. In this extent, the development of new drugs for tuberculosis treatment is a field that is gaining importance. The 2(quinolin-4-yloxy) acetamides are molecules that showed promising bactericidal effects in Mycobacterium tuberculosis, which motivated us to continue studying to improve their mycobactericidal activity and also perform their chemical and biological characterization. In order to continue the 2(quinolin-4-yloxy) acetamides derivatives development, the target identification of these molecules is a keystone and is also the focus of this study. Initially, it was hypothesized that DNA gyrase was their molecular target due to the great chemical similarities between the 2(quinolin-4-yloxy) acetamides and the fluoroquinolones. Despite our results that the 2(quinolin-4-yloxy) acetamides have diminished effects in ofloxacin resistant clinical isolates, the results obtained with the gyrA point mutant and with DNA gyrase protein revealed that this enzyme is not the molecular target of these compounds. The new target identification strategy involved the selection of spontaneous mutants for our lead compound 12L, characterization of this mutant strain against other 2(quinolin-4-yloxy) acetamides derivates and whole genome sequencing. Whole genome sequencing data allowed the identification of a single mutation (T313A) in the QcrB protein, which is the B subunit of cytochrome bc1 complex. This mutation was confirmed by Sanger sequencing and molecular docking results confirmed the importance of this residue for proteindrug interaction. The cytochrome bc1 complex is involved in the electron transport of the bacilli?s respiratory chain, and therefore it appears to be an interesting target, especially to treat the latent form of the disease. We hope that this work contributes to the 2(quinolin-4-yloxy) acetamides development for tuberculosis treatment. / A alta incid?ncia da tuberculose, em ?mbito mundial, ? de grande preocupa??o. Para combater esta doen?a, diferentes estrat?gias v?m sendo desenvolvidas pela Organiza??o Mundial da Sa?de (OMS). Dentre os pilares que comp?em a End TB strategy, podemos destacar a pesquisa intensiva e a inova??o. Neste ?mbito, o desenvolvimento de novos f?rmacos para tuberculose vem ganhando destaque. As 2(quinolin-4-il?xi) acetamidas s?o mol?culas que demonstraram resultados bactericidas promissores frente ao Mycobacterium tuberculosis, o que nos motivou a realizar novos estudos para melhorar a atividade e realizar a caracteriza??o qu?mica e biol?gica destas mol?culas. A fim de continuar o desenvolvimento da s?rie quinol?nica, a identifica??o do seu alvo molecular foi o foco deste trabalho. Inicialmente, levantou-se a hip?tese de que a DNA girase seria o alvo molecular, uma vez que as 2(quinolin- 5-il?xi) acetamidas possuem grande similaridade estrutural com as fluoroquinolonas. Apesar das 2(quinolin-4-il?xi) acetamidas terem apresentado menor atividade frente a isolados cl?nicos resistentes a ofloxacino, os resultados de atividade obtidos frente a uma mutante pontual de gyrA e frente ? prote?na indicam que a DNA girase n?o ? o alvo destas mol?culas. A nova estrat?gia para identifica??o de alvo envolveu a sele??o de mutantes espont?neos para o composto l?der 12L, caracteriza??o desta cepa frente aos demais compostos da s?rie e sequencimento total do genoma. Este permitiu a identifica??o de uma ?nica muta??o (T313A) localizada na prote?na QcrB, que ? a subunidade B do complexo citocromo bc1. Esta muta??o foi confirmada por sequenciamento de Sanger e o docking molecular aferiu a import?ncia deste res?duo na intera??o prote?na-composto. O complexo citocromo bc1 est? envolvido no transporte de el?trons na cadeia respirat?ria do bacilo, e por isso ? um alvo molecular interessante, principalmente para combater a forma latente da doen?a. Esperamos que este trabalho contribua no processo de desenvolvimento das 2(quinolin-4-il?xi) acetamidas para o tratamento da tuberculose.