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| Author(s): |
Juliana Maria de Lima
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2018-05-11 |
| Examining board members: |
Carmen Lúcia Cardoso;
Emanuel Carrilho;
Quezia Bezerra Cass;
Maria Cristina Nonato;
Anderson Rodrigo Moraes de Oliveira
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| Advisor: | Carmen Lúcia Cardoso |
| Abstract | |
Enzymes play a key physiological role in organisms, either under normal and pathological conditions, which make them attractive targets for therapeutic interventions. Small molecules capable to inhibit specific target enzymes are used for the treatment of several inflammatory diseases, cancer, AIDS, among others. However, to identify small inhibitory molecules is still a limiting step in drugs discovery. In this context, the improvement and development of robust and reliable novel assays to accelerate the discovery and characterization of new inhibitors have become extremely relevant. This study describes an appropriate direct method to quantify the enzymatic activity of Nucleoside Diphosphate Kinase B (NDKb) from human (NME2) and Leishmania major (LmNDKb). It can be applied to free enzyme in solution or immobilized enzyme into silica capillary (ICER). The separation of both the substrates and products was achieved using ion-pair chromatography, it can be applied to direct quantification of free NDKb activity, method 1D-LC-UV. In order to quantify the activity of the immobilized enzymes, an on-line ICER-LC-UV method was developed. Initially, the enzymatic catalysis occurred into the ICER. Sequentially, the reaction mixture was transferred to an analytical column, where analytes were separated. From this method, it was possible to determine the optimum pH and kinetic parameters for the immobilized enzymes. Well stablished NDKb inhibitors with different strenght and mechanisms of action were used to modulate the on-line ICER-LC-UV method as an inhibitor screening assay. (-)-Epicatechin gallate was used as proof of concept in order to validate the application of the on-line ICER-LC-UV method to identify and characterize the target\'s enzymes NME2 and LmNDKb inhibitors. In summary, the direct quantification method coupled to the immobilized enzymes increases the likelihood of identifying the enzymatic activity and screening of inhibitors of the target enzymes when compared methods well described in the literature. (AU) | |
| FAPESP's process: | 14/06907-0 - Immobilization nucleoside diphosphate kinase (NDK) enzyme Leishmania major: the study of conditions for the application in the screening of ligands |
| Grantee: | Juliana Maria de Lima |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |