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Master's Dissertation
DOI
https://doi.org/10.11606/D.75.2022.tde-17082022-165601
Document
Author
Full name
Rodrigo Cendron
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2022
Supervisor
Committee
Montanari, Carlos Alberto (President)
Murakami, Mario Tyago
Sousa, Clarisa Beatriz Palatnik de
Title in Portuguese
Estudo da cinética enzimática entre potenciais fármacos e cisteína-proteases por fluorimetria
Keywords in Portuguese
cinética enzimática
cisteína proteases
doença de Chagas
fluorescência
Leishmaniose
Abstract in Portuguese
O presente trabalho se dedica ao estudo da atividade inibitória de algumas moléculas orgânicas, desenvolvidas com o intuito de atuarem como fármacos, e seis enzimas da família das cisteíno proteases, empregando fluorimetria como método analítico. Estas atividades estão inseridas no contexto da busca de novos fármacos para o tratamento de diversas doenças, dentre as quais se destacam o Mal de Chagas e a Leishmaniose, que estão relacionados respectivamente aos ensaios com as enzimas Cruzaína e CPB. As cisteína proteases da família da papaína encontradas nos parasitas causadores destas doenças foram identificadas como essenciais para o seu ciclo de vida e, portanto, servem como alvos moleculares validados, pois a sua inibição poderia levar à morte seletiva do agente patogênico. Realizou-se também a expressão e a purificação da enzima Cruzaína, com o intuito de viabilizar os estudos com elas. As dipeptidilnitrilas têm sido estudadas nas últimas décadas como inibidores covalentes reversíveis de enzimas proteolíticas do tipo da papaína, com atuação seletiva e potente; juntamente a essa classe de substâncias, iniciou-se os testes com peptóides, que, apesar de serem uma classe inédita de substâncias apresentaram atividade inibitória. Neste trabalho, são empregadas técnicas analíticas com foco principal em medidas fluorimétricas, com o objetivo de estudar os efeitos das substâncias de interesse sobre os alvos moleculares. Os testes realizados com as Catepsinas L, K, S e B estão correlacionados à busca de inibidores com possível atividade farmacêutica sobre muitas enfermidades autoimunes e diversos tipos de câncer. No entanto, é importante mencionar que os estudos estão limitados aos estágios iniciais da pesquisa para uma substância bioativa inédita, etapa em que são realizados os ensaios in vitro dos possíveis fármacos com o alvo molecular, para tanto, serão empregadas técnicas fluorimétricas para o estudo da cinética de inibição enzimática, buscando determinar a capacidade inibitória dos possíveis fármacos e, também, extrair informações sobre os alvos moleculares através da busca da relação entre a estrutura química e a atividade dos inibidores, contribuindo, assim, para o desenvolvimento da pesquisa de forma mais geral.
Title in English
Study of enzymatic enzyme between potential drugs and cysteines by fluorimetry
Keywords in English
Chagas disease
cysteine proteases
enzymatic kinetics
fluorescence
Leishmaniasis
Abstract in English
This work is dedicated to the study of inhibitory activity among some organic molecules developed with the intention of acting as drugs and six enzymes of the cysteine proteases family employing fluorimetry as analytical method. These activities are inserted in the context of the search for new drugs for the treatment of several diseases, among which stand out the Chagas disease and leishmaniasis, which are related respectively to the assays with the enzymes Cruzain and CPB. Papain family cysteine proteases found in parasites causing these diseases have been identified as essential to their life cycle and therefore serve as validated molecular targets, as their inhibition could lead to selective death of the pathogen. In association with the analytical activity with these enzymes, the expression and purification of the enzymes CPB and Cruzaína was performed in order to enable the studies with these enzymes. Dipeptidilnitriles have been studied in recent decades as reversible covalent inhibitors of papain-type proteolytic enzymes with selective and potent activity, along with this class of substances started tests with peptides, that despite being an unprecedented class of substances have shown inhibitory activity. In this work, analytical techniques are used with a main focus on fluorimetric measurements in order to study the effects of substances of interest on molecular targets. The tests performed with Cathepsins L, K, S and B are correlated to the search for inhibitors with possible pharmaceutical activity on a large number of autoimmune diseases and several types of cancer, however, it is important to mention that the studies are limited to the initial stages of the research for an unprecedented bioactive substance, a stage in which the in vitro trials of possible drugs with the molecular target are performed, whereas fluorimetric techniques will be used for the study of enzyme inhibition kinetics, seeking to determine the inhibitory capacity of possible drugs and also extract information on molecular targets through the search for the relationship between the chemical structure and the activity of inhibitors, thus contributing to the development of research.
 
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Publishing Date
2022-08-19
 
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