Glucose 6-phosphate dehydrogenase as molecular target for drug development: structural studies and inhibitors identification

Author(s)

Gustavo Fernando Mercaldi

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-07-13

Examining board members

Artur Torres Cordeiro; Alisson Campos Cardoso; Marcio Vinicius Bertacine Dias; Paulo Sérgio Lopes de Oliveira; Ariel Mariano Silber

• Advisor: Artur Torres Cordeiro

Abstract

American trypanosomiasis and leishmaniasis are life-threatening infectious diseases, caused by flagellate protozoans Trypanosoma cruzi e Leishmania spp, respectively. Affecting millions of individuals, mainly in developing countries of tropical and subtropical areas, those conditions have a great impact in affected populations. Currently available drugs to treat those diseases suffer from limitations related to security, efficacy or treatment regimens. This scenario highlights the relevance of initiatives that contributes to the comprehension of the parasites biology or to the development of new clinically useful drugs. The enzyme glucose-6-phosphate dehydrogenase (G6PDH) has been explored as a promising target for the development of new drugs. G6PDH catalysis the first and rate limiting step of the pentose phosphate pathway, which is essential for the maintenance of oxidative balance that also provides cells with important molecules for the biosynthesis of lipids, nucleotides, vitamins and amino acids. This enzyme is essential for the survival of protozoans from the genus Trypanosoma, in such a way that depletion of protein levels, or its specific inhibition by organic molecules, hampers parasites growth. Presently, the best G6PDH inhibitors are halogenated derivatives of the steroid dehydroepiandrosterone (DHEA), capable to strongly inhibit G6PDHs from humans (HsG6PDH) and T. cruzi (TcG6PDH), but with no effect on the L. mexicana enzyme (LmG6PDH). New G6PDH inibitors have been identified in a 30.000 compounds library by a high throughput screening (HTS) campaign with the TcG6PDH. Quinazolinone and thienopyrimidine derivatives showed to inhibit the TcG6PDH through an uncompetitive mechanism and preliminary structure-activity relationship were established for both inhibitor classes. Structural features that contribute for selectivity towards TcG6PDH over the human enzyme were identified and experiments with T. cruzi epimastigote form showed that quinazolinone derivatives arrest parasite growth in vitro, suggesting that those molecules are promissing inhibitors that should be considered for further optimization. Quinazolinone derivatives also showed to inhibit the LmG6PDH by uncompetitive mechanism, supporting the notion that proteins from Leishmania have an interaction site for inhibitors upon formation of an enzyme-substrate(s) complex. Quinazolinones derivatives with activity over the LmG6PDH emerge as potential chemical probes to evaluate the importance of this enzyme for parasites from the genus Leishmania. Finally, structural studies with G6PDHs allowed the obtention of the crystallographic structure from the ternary complex Tc?G6PDH-G6P-NADPH, which reveals details on the molecular basis of substrate and cofactor recognition. Comparison of this structure with the human homologous enzyme revealed a cavity on the vicinity of the cofactor-binding site that might be explored towards the design of new competitive inhibitors selective for the TcG6PDH. In this way, our results contribute to improve the structural and functional knowledge about the G6PDHs, an enzyme with an essential biological role, and may ultimately help to better understand the trypanosomatids parasite biology and to development of new therapeutic alternatives that satisfy unmet medical needs for Chagas disease and leishmaniasis (AU)

• FAPESP's process: 10/17849-0 - Development of a High Throughput Screening of inhibitors for the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi
• Grantee: Gustavo Fernando Mercaldi
• Support Opportunities: Scholarships in Brazil - Doctorate