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Master's Dissertation
DOI
10.11606/D.75.2007.tde-25102007-114804
Document
Author
Full name
Hélton José Wiggers
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2007
Supervisor
Committee
Montanari, Carlos Alberto (President)
Loh, Watson
Silva, Alberico Borges Ferreira da
Title in Portuguese
Planejamento de inibidores da enzima gliceraldeído-3-fosfato desidrogenase de Trypanosoma cruzi por biocalorimetria
Keywords in Portuguese
biocalorimetria
Doença de Chagas
enzima
Abstract in Portuguese
A identificação de compostos bioativos que atuem em proteínas é imprescindível no conceito atual da Química Medicinal. Quase todos os processos biossintéticos dos organismos vivos são regulados por enzimas. Dentro deste contexto a enzima glicossomal Gliceraldeído-3-Fosfato desidrogenase (GAPDH) do Tripanosoma cruzi (agente etiológico causador da Doença de Chagas) tem-se mostrado um alvo promissor para o planejamento de inibidores contra o parasito. A Calorimetria de Titulação Isotérmica (ITC) é uma ferramenta robusta na identificação e otimização de compostos bioativos. Através da ITC podem-se determinar parâmetros cinéticos da atividade enzimática (kcat, KM e Ki) e parâmetros termodinâmicos de interação (DH, DG e DS) entre o alvo biomacromolecular e o composto bioativo. Neste trabalho desenvolveu-se um protocolo de ensaio cinético para a enzima GAPDH através da ITC, visando à identificação de novos compostos tripanossomicidas. A atividade da enzima foi avaliada na presença de diferentes concentrações (0 a 10 % v/v) de dois solventes orgânicos padrões (dimetilsulfóxido e metanol). Esses solventes são utilizados em bioensaios e também para aumentar a solubilidade de substâncias em água. A concentração de 5 % v/v de ambos solventes orgânicos foi determinada como aquela em que a GAPDH manteve-se estável. Houve um aumento significativo da atividade enzimática em relação aos experimentos sem uso de co-solventes. Através do ensaio padronizado foram testados os seguintes compostos frente à GAPDH: ácido 4-butilfenil-amino-metileno-fosfônico, mangiferina, quercetina-3-O- B-L-arabinopiranosídeo, caempferol-3-O-alfa-D-(6-O-p-coumaroyl) glucosídeo e marcianina. As constantes de inibição (Ki) dessas substâncias apresentam-se no intervalo de 20,1 (±1,70) a 27,3 (±2,47) mM. Uma nova classe de pequenas moléculas de origem em produtos naturais e denominada xantonas foi identificada com potencial atividade tripanossomicida.
Title in English
Structure-based inhibitor design of the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase enzyme using isothermal titration calorimetry
Keywords in English
biocalorimetry
Chagas´ disease
enzyme kinetcs
Abstract in English
The identification of new bioactive chemical samples that act on proteins is essential in modern Medicinal Chemistry. Enzymes regulate almost all-biosinthetic processes in the life of an organism. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas´ disease. Its glycosomal Glyceldehide-3-Phosphate Dehydrogenase enzyme (GAPDH) is regarded as a promising target for the structure-based discovery and development of new antiparasitic drugs. The Isothermal Titration Calorimetry (ITC) is a powerful tool in the discovery and optimization of new bioactive substances. The use of ultrasensitive calorimetry for the life sciences via isothermal titration calorimetry provides a new paradigm in the drug discovery area. It greatly improves the chances of producing a probe molecule that binds to the target enzyme. Kinetics (kcat, KM and Ki) and thermodynamics parameters (DH, DG and DS) can be determined by measuring the interactions between the target enzyme and bioactive chemical substances. In this work, a protocol for a kinetic enzyme assay was developed to study the activity of T. cruzi GAPDH using ITC. The enzyme activity in the presence of different standard co-solvent concentrations (0-10 % v/v), used in bioassays, was evaluated. The concentration of 5 % v/v for both co-solvents was chosen for standardized condition. This was elected so due to a great increase in the enzyme activity when compared to the experiments with no co-solvent added. Using the standardized enzyme assay protocol the following chemical substances were tested against the T. cruzi GAPDH: 4-butylfenyl-amine-methylenephosphonic acid, quercetin-3-O-B-L-arabinopyranoside, mangiferin, kaempferol-3-O- alfa-D-(6-O-p-coumaroyl) glucoside and martianine. The inhibition constants (Ki) were measured for these substances and their values are in the range of 20,1 (±1,7 ) and 27,3 (±2,5) mM. A natural product molecular class named xantone was identified as a new potential trypanocide agent.
 
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Publishing Date
2007-10-29
 
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