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Master's Dissertation
DOI
10.11606/D.59.2014.tde-21072014-144130
Document
Author
Full name
Felipe Antunes Calil
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2014
Supervisor
Committee
Cardoso, Carmen Lúcia (President)
Fietto, Juliana Lopes Rangel
Moraes, Marcela Cristina de
Title in Portuguese
Biorreatores capilares de NTPDase-1 de Trypanosoma cruzi: desenvolvimento e aplicação na triagem de inibidores seletivos
Keywords in Portuguese
Ensaios enzimáticos
Expressão heteróloga
Imobilização de enzimas
NTPDase-1 T.cruzi
Abstract in Portuguese
Uma das estratégias utilizadas no desenvolvimento de novas drogas envolve a descoberta de compostos que modulem a atividade de enzimas, importantes no processo infeccioso de patógenos. Uma abordagem interessante na triagem de novos ligantes é o uso de métodos baseados na imobilização de enzimas em suportes cromatográficos acoplados a sistemas de cromatografia líquida. O uso de IMERs (Immobilized Enzyme Reactors) como uma fase estacionária acoplado a sistemas de cromatografia líquida de alta eficiência consiste em uma estratégia para triagem de compostos rápida e eficiente e tem vantagens em relação ao uso de enzimas em solução. A enzima NTPDase-1 de Trypanosoma cruzi age como um facilitador da infecção do patógeno, inibindo assim a resposta imune do hospedeiro, permitindo uma infecção silenciosa, o que sugere seu uso como um bom alvo na busca por inibidores. Neste trabalho, a enzima NTPDase-1 foi imobilizada na parede interna de capilares de sílica fundida formando ICERs (Immobilized Capillary Enzyme Reactors). Estudos das condições de uso destes biorreatores juntamente com o desenvolvimento de um método cromatográfico multidimensional, foram realizados e validados. A otimização do método cromatográfico e sua validação, apresentaram ótimos resultados em relação aos valores obtidos para os parâmetros avaliados para métodos bioanalíticos. A imobilização da enzima foi realizada com sucesso, sendo possível a detecção da atividade catalítica no sistema cromatográfico (TcNTPDase1-ICER). Foi realizado também, o estudo cinético para ATP no TcNTPDase1-ICER, obtendo-se KM de 0,317 ± 0,044 mM, que comparado com estudos em solução, KM de 0,096 mM, ainda apresenta grande afinidade pelo substrato.
Title in English
Capillary bioreactors of NTPDase-1 Trypanosoma cruzi: Development and application in the selective inhibitors screening 2014.
Keywords in English
Enzymatic assays.
Heterologous expression
Immobilization of enzymes
NTPDase-1 T.cruzi
Abstract in English
One of the strategies used in the development of new drugs involves the discovery of compounds that modulate the activity of enzymes, important in the infectious pathogens process. An interesting approach in the screening of new ligands is the use of methods based on immobilization of enzymes in chromatographic supports coupled to liquid chromatography systems. The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems consist in a strategy to a fast and efficient compounds screening and it has advantages comparing to the use of enzymes in solution. The enzyme NTPDase-1 Trypanosoma cruzi acts as a pathogen infection facilitator, thus inhibits the host immune response allowing a silent infection, suggesting its use as a good target in the search for inhibitors. In this paper, the enzyme NTPDase-1 was immobilized for the manufacturing of ICERs (Immobilized Capillary Enzyme Reactors). Studies of conditions to the use of these bioreactors in the ligands screening along with the development of a multidimensional chromatographic method were performed and validated. The chromatographic method optimization and validation, presented excellent results, relating to the obtained values, from evaluated parameters in bioanalytical methods. The enzyme immobilization was successfully performed, being possible to detect the catalytic activity in the chromatographic system (TcNTPDase1-ICER). The kinetic study for the substrate ATP was also performed in the TcNTPDase1-ICER, obtaining KM of 0.317 ± 0.044 mM, which in comparison with studies in solution KM of 0.096 mM, still presents high affinity for the substrate.
 
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Release Date
2018-07-21
Publishing Date
2014-10-29
 
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