Diabetes Type II Regulation – Inhibitory Potential of hlGP with Different Natural Compounds

• Autor(a) principal: Lopes, Filipa Alexandra Afonso
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/117534
• topic: Type II Diabetes; Glycogen Phosphorylase; Inhibition; MicroScale Thermophoresis; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Diabetes melitus is a metabolic disease characterized by a lack of insulin production and/or increased cellular resistance to insulin, leading to high levels of glucose in bloodstream (hyperglycaemia). Glycogen phosphorylase (GP) is a key enzyme involved in the cascade responsible for the breakdown of glycogen to glucose-1-phosphate. GP is a validated target for the treatment of diabetes as some GP-specific inhibitors lower blood glucose levels in vivo. A computational study carried out by our collaborators at the Faculty of Sciences, University of Porto, has identified possible compounds with high inhibitory capacity for GP. The main aim of this dissertation was to follow up these compounds and experimentally validate the theoretical data. To achieve this, the first objective was the optimization of the heterologous expression and purification of human liver GP to obtain the recombinant protein in its less active form (GPb), and in its most active form (GPa) through phosphorylation. Several thermal shift assays (TSA) were carried out and were decisive in choosing the best conditions for the stabilization of both proteins. Microscale Thermophoresis (MST) was applied to quantify the interactions of human GPb and several compounds. Dissociation constants, Kd, were determined for the flavonoids genistein (1.2 ± 0.4 µM), 8-prenylnaringenin (4.1 ± 1.2 µM) and 8-prenylgenistein (7.4 ± 3.7 µM). Overall, these results agree with the theoretical data calculated by Molecular Dynamics and Free-energy perturbations, thus validating the binding affinity of GP for these compounds. Kinetic assays to determine the enzyme activity are ongoing, but preliminary assays with the two forms of the enzyme showed that GPa has a high activity while GPb did not show activity. An assay with GPa and caffeine (inhibitor) showed reduced enzyme activity. These results have shown to be quite promising and it is intended in the future to determine the parameters V0, Vm and Km of the reaction, and to determine the protein inhibition constants with the different inhibitors to follow up the MST data. The combined theoretical and experimental data suggested the flavonoids genistein, 8PN and 8PG as potential inhibitory compounds for GP and may, in the future, be explored for the treatment of diabetes.