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Previous issue date: 2012-01-18 / Tuberculosis re-emerged in the mid 80s and currently about two million people die each year due to this disease. The resurgence of tuberculosis has become a threat to public health. The high susceptibility of HIV-infected patients and the proliferation of multi-drug resistant strains have created the need to develop new therapies. However, no new class of drugs against tuberculosis was developed in the last 45 years. Thus, it becomes imminent need to develop new antituberculosis agents. As purine metabolism could be implicated in mycobacterial latency the purine nucleoside phosphorylase (EC 2.4.2.1) has been proposed as target for development of antibacterial drugs. PNP catalyzes the reversible cleavage in the presence of inorganic phosphate of N-ribosidic bonds of the purine nucleosides and deoxynucleosides, except adenosine. This reaction generates the purine base and ribose (deoxyribose)-1-phosphate. PNP is specific for purine nucleosides in the β-configuration and cleaves the glycosidic bond with inversion of configuration to produce α-ribose-1-phosphate. In this work PNP was crystallized in association with inosine, hypoxanthine and acyclovir, substrate, product and inhibitor, respectively, and data were collected using synchrotron radiation. In addition, molecular dynamics simulations and isothermal titration calorimetry experiments were used to provide detailed information on the dynamic properties and to investigate the profile and thermodynamic affinities of MtPNP associated with these molecules. With obtained results we hope to contribute to the search of new selective inhibitors for MtPNP, since differences between the mycobacterial and human enzyme binding sites have been also identified, making structure-based drug design feasible. / A tuberculose ressurgiu na metade dos anos 80 e, atualmente aproximadamente dois milh?es de pessoas morrem por ano devido a esta doen?a. O ressurgimento da tuberculose tornou-se uma amea?a ? sa?de p?blica. A alta susceptibilidade dos pacientes infectados com HIV e a prolifera??o de cepas resistentes a m?ltiplas drogas t?m criado a necessidade de se desenvolver novas terapias. No entanto, nenhuma nova classe de drogas contra a tuberculose foi desenvolvida nos ?ltimos 45 anos. Deste modo, torna-se iminente a necessidade de se desenvolver novos agentes antituberculose. Como o metabolismo de purinas pode estar implicado na lat?ncia da micobact?ria, a purina nucleos?deo fosforilase de Mycobacterium tuberculosis (MtPNP) (EC 2.4.2.1) foi proposta como alvo para o desenvolvimento de drogas antibacterianas. A PNP catalisa a clivagem revers?vel, na presen?a de fosfato inorg?nico, de liga??es N-ribos?dicas de nucleos?deos e desoxinucleos?deos de purina, com exce??o da adenosina. Esta rea??o produz base p?rica livre e ribose (desoxiribose)-1-fosfato. A rea??o se processa com invers?o de configura??o, de β-nucleos?deos para α-ribose-1-fosfato. Neste trabalho a PNP foi cristalizada em associa??o com inosina, hipoxantina e aciclovir, substrato, produto e inibidor, respectivamente, e os dados foram coletados utilizando radia??o s?ncrotron. Al?m disso, a simula??o por din?mica molecular e experimentos de calorimetria por titula??o isot?rmica foram utilizadas para fornecer informa??es detalhadas acerca das propriedades din?micas, e investigar o perfil termodin?mico e afinidades da MtPNP associada com estas mol?culas. Com os resultados obtidos n?s esperamos contribuir para a busca de novos inibidores seletivos de MtPNP, j? que as diferen?as entre os s?tios de liga??o da enzima humana e de micobact?ria foram identificadas , tornando vi?veis projetos de desenho de drogas baseados na estrutura.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede2.pucrs.br:tede/1674 |
| Date | 18 January 2012 |
| Creators | Caceres, Rafael Andrade |
| Contributors | Azevedo Junior, Walter Filgueira de |
| Publisher | Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de, PUCRS, BR, Faculdade de Medicina |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS, instname:Pontifícia Universidade Católica do Rio Grande do Sul, instacron:PUC_RS |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 7620745074616285884, 500, 600, -8624664729441623247 |
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