Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-10-01T18:36:32Z No. of bitstreams: 1 475353 - Texto Completo.pdf: 4655532 bytes, checksum: c5dbc2135d9425bffb99fb72fd2f27dd (MD5) / Made available in DSpace on 2015-10-01T18:36:32Z (GMT). No. of bitstreams: 1 475353 - Texto Completo.pdf: 4655532 bytes, checksum: c5dbc2135d9425bffb99fb72fd2f27dd (MD5) Previous issue date: 2015-06-29 / the second responsible for most of the deaths worldwide. The latest estimates of WHO showed that 9 millions of new cases of TB occurred in 2013 and 1.5 million of deaths. The emergence of new drug resistant M. tuberculosis strains is becoming a serious increasing problem as the treatment of infected patients with multi drug-resistant TB and extensively drug-resistant TB strains is much more difficult and costly. This brings discussions about the drastic situation of virtually untreatable TB cases and shows the urgent need to introduce new and effective anti-TB drugs. The research for the development of new antimycobacterial agents becomes a necessity, as well as the identification of new targets for future drugs. The histidine biosynthetic pathway is comprised of ten enzyme steps catalyzed by eight enzymes. This pathway is present in prokaryotic organisms, lower eukaryotic organisms and plants, but is absent in animals, which is in accordance with the principles of selective toxicity. Mutagenesis studies have shown that the genes of this pathway are essential for the survival of M. tuberculosis. The histidinol dehydrogenase enzyme, encoded by hisD, performs the last two steps in the biosynthesis of histidine, converting L-histidinol to L-histidine. The essentiality of the hisD gene in M. tuberculosis mutants with referred gene knockout is already described in the literature. This work describes kinetic studies using thermodynamic parameters, fluorescence spectroscopy, and pre-stationary states to better understand the enzymatic mechanism of MtHisD. Characterization of the reaction catalyzed by mycobacterial HisD is important to structure-based drug development. The data from enzyme?s kinetic characterization were the starting point for HisD specific inhibitors planning, selection, and testing. A series of eleven hydrazones derived from L-histidine was synthesized, from which four compounds were identified as showing a competitive inhibition profile for L-histidinol substrate. The interactions of these compounds with the enzyme were analyzed by molecular docking to understand the inhibitory mechanism. Results from this work are believed to enhance the understanding of mycobacterial histidine metabolism. Moreover, data from inhibition studies with the synthesized compounds, serve as the starting point for the development of new molecules to enhance the enzyme inhibition and to inhibit the M. tuberculosis growth. / Dentre as doen?as infecciosas que acompanham o homem ao longo da hist?ria, a tuberculose (TB), atualmente, ? a segunda respons?vel pelo maior n?mero de mortes no mundo. As ?ltimas estimativas da Organiza??o Mundial da Sa?de apontam 9 milh?es de novos casos de TB em 2013 e 1,5 milh?es de mortes. O surgimento de novas linhagens de M. tuberculosis resistentes aos f?rmacos utilizados no tratamento est? se tornando um problema s?rio e crescente, uma vez que o tratamento de pacientes infectados com cepas de tuberculose resistente ? m?ltiplos f?rmacos e extensivamente resistente ? f?rmacos ? muito mais dif?cil e oneroso. Isso remete a discuss?es sobre a dr?stica situa??o de casos de TB virtualmente incur?veis e aponta para a urgente necessidade de introduzir novos e eficazes medicamentos anti-TB no mercado. A pesquisa para o desenvolvimento de novos agentes antimicobacterianos torna-se necess?ria, bem como a identifica??o de novos alvos para futuros medicamentos. A via de bioss?ntese de histidina compreende dez etapas enzim?ticas catalisadas por oito enzimas. Esta via est? presente nos organismos procari?ticos, organismos eucari?ticos inferiores e em plantas, mas est? ausente em animais, corroborando com os princ?pios de toxicidade seletiva. Estudos de mutag?nese demonstraram que os genes desta via s?o essenciais para a sobreviv?ncia do M. tuberculosis. A enzima histidinol desidrogenase, codificada pelo gene hisD, catalisa a ?ltima etapa da bioss?ntese de histidina, convertendo L-histidinol para L-histidina. A essencialidade do gene hisD em mutantes de M. tuberculosis, com o referido gene nocauteado, j? est? descrita na literatura. Este trabalho trata sobre o aprofundamento dos estudos cin?ticos, utilizando ensaios para determina??o de par?metros termodin?micos, fluorimetria e ensaios em estado pr?estacion?rio. A caracteriza??o da rea??o catalisada pela enzima HisD micobacteriana ? uma etapa importante para o desenvolvimento de novos f?rmacos de a??o espec?fica que permitam o melhor controle da tuberculose. Os dados de caracteriza??o cin?tica da enzima foram utilizados como ponto de partida para o planejamento, sele??o e teste de inibidores seletivos contra a HisD de M. tuberculosis. Uma s?rie de onze hidrazonas derivadas da Lhistidina foi sintetizada, foram identificados quatro compostos com perfil de inibi??o competitiva pelo substrato L-histidinol. As intera??es destes compostos com a enzima foram analisadas por docagem molecular para melhor compreens?o do mecanismo inibit?rio. Os resultados deste trabalho colaboram ainda para uma melhor compreens?o do metabolismo da bioss?ntese de histidina em micobact?rias. Al?m disso, os dados dos estudos de inibi??o com os compostos aqui sintetizados servem como ponto de partida para o desenvolvimento de novas mol?culas visando a otimiza??o da inibi??o da enzima e a da inibi??o do crescimento do M. tuberculosis.

Made available in DSpace on 2014-12-17T14:03:27Z (GMT). No. of bitstreams: 1 CarlosEMG.pdf: 434119 bytes, checksum: cffd1dddf77ec049b23dadc1bfa89773 (MD5) Previous issue date: 2004-12-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50?C, and at 100?C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae / Um inibidor de proteinase foi isolado de sementes de Crotalaria pallida por fracionamento com sulfato de am?nio, cromatografia de afinidade em Sepharose com tripsina acoplada e precipita??o com ?cido tricloroac?tico (TCA). O inibidor de tripsina, denominado ITC, possui massa molecular de 32,5 kDa determinada por SDS-PAGE e ? composto por duas subunidades, uma de 25,7 e outra de 5,6 kDa, ligadas por pontes dissulfeto; uma caracter?stica t?pica da fam?lia de inibidores de Kunitz. ITC foi est?vel at? 50?C e a 100?C sua atividade residual foi cerca de 60%. ITC tamb?m foi est?vel em valores de pHs variando de 2 a 12. A inibi??o da tripsina por ITC ? do tipo n?o-competitivo, com um Ki de 8,8 x 10-7M. ITC inibe fracamente outras proteinases ser?nicas como quimotripsina e elastase. A inibi??o da papa?na (44%), uma proteinase ciste?nica, foi indicativa da bifuncionalidade de ITC. Ensaios in vitro contra proteinases digestivas de v?rios insetos praga das ordens Lepidoptera, Diptera e Coleoptera foram realizados. ITC inibiu em cerca de 100% enzimas digestivas de Ceratitis capitata (mosca-das-frutas), Spodoptera frugiperda (lagarta do cartucho do algod?o), e Alabama argillacea (curuquer? do algod?o). ITC tamb?m inibiu em 74,4% enzimas digestivas de Callosobruchus maculatus (broca do feij?o) uma praga da ordem Coleoptera. ITC, quando adicionado a dietas artificiais, afetou fracamente o desenvolvimento de larvas de C. capitata e teve um WD50 de 2,65% contra larvas de C. maculatus