Planejamento, síntese e avaliação da atividade biológica de potenciais inibidores da enzima trans-sialidase de Trypanosoma cruzi

Abstract

DE ANDRADE, P. Design, synthesis and biological activity evaluation of potential inhibitors of the Trypanosoma cruzi trans-sialidase enzyme. 2012. 183 f. Thesis (Doctoral). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto, 2012.Chagas' disease, also known as American trypanosomiasis, is one of the most devastating tropical diseases and it is caused by the protozoan Trypanosoma cruzi. The parasite expresses a cell surface enzyme, Trypanosoma cruzi trans-sialidase (TcTS), responsible for the transference of sialic acids from host cells to terminal β-galactose molecules present on its glycoprotein surface. The sialylated glycoprotein molecules are involved in the attachment and subsequent penetration of the parasite into host cells. As a result, TcTS plays a key role in the recognition and invasion of host cells. Moreover, the lack of trans-sialidase in humans makes TcTS a potential drug target to be explored, however no strong inhibitors (at nanomolar range) of this enzyme are known to date. Considering the importance of galactose unit and the carboxyl function in sialic acid for interactions in the active site of TcTS, we have focused on the synthesis of galactose derivatives containing succinic acid in different positions of the sugar ring and on their biological evaluation against TcTS. In addition, we have proposed the search for new TcTS inhibitors applying molecular modeling techniques, like virtual screening based on the enzyme´s active site. Commercially available α,β-D-galactose and methyl-β-D-galactopyranoside were treated with suitable reagents to afford some intermediates with just one free hydroxyl group in few steps. Treatment of these compounds with succinic anhydride in pyridine afforded derivatives thereof with succinic acid in all galactosidic ring positions. After deprotection step it was only obtained the derivative containing succinic acid at position 4 (85), which was tested in in vitro TcTS fluorimetric assay for evaluation of its inhibitory activity. In Addition, 85 was tested in in vitro assays for assessment of their trypanocidal activity and cytotoxic. According to the biological assays, compound 85 showed promising inhibitory activity (56%) at 1.0 mM concentration. This preliminary result was important both to show that this type of derivative can act as an inhibitor of TcTS and to guide the synthesis of new derivatives of galactose. The trypanocidal activity value was lower than 40% (0.5 mM) and there was no cytotoxicity at 0.5 mM concentration. The virtual screening studies performed in this work through docking simulations resulted in the selection of the 50 top-ranked molecules, based in the highest score orientation, among 50.000 found at diverset data base. The next step involves new studies to filter the most promising molecules to be tested against TcTS.

Avaliação do impacto de uma enzima modificada capaz de reconhecer glicanas α-2,3-sialiladas na evolução da infecção experimental por Trypanosoma cruzi

“…Essa atividade enzimática da TS nativa, pode ser observada em um ensaio enzimático com a utilização de TS recombinante e proteínas ricas em ácido siálico como a fetuína, sendo capaz de demonstrar a eficiência ao reproduzir os feitos da TS nativa (AMAYA et al, 2004;DAMAGER et al, 2008;LANTOS et al, 2016;ZINGALES et al, 1987). Considerando essa alta especificidade ao ácido siálico, vários estudos desenvolvem ferramentas direcionadas ao ácido siálico ou à proteína TS para inibição do processo de invasão e, portanto, com potencial ação anti-T.cruzi (AGUSTI; GALLO-RODRIGUEZ; ANDRADE, 2012;KASHIF et al, 2017;LARA-RAMIREZ et al, 2017). Por exemplo a proteína recombinante "SiaFind™α2,3-Specific Lectenz®", do tipo lectina com alta afinidade e especificidade ao ácido siálico na ligação α2,3.…”