Estudos alvo-direcionados de produtos naturais de actinobactérias marinhas

Abstract

The development of different strategies for prospecting natural products which have the molecular target as the key perspective has contributed to innovation and discovery of new substances with anticancer potential. In this context, this work carried out the study of natural products from marine actinobacteria through different approaches for the elucidation of the molecular target of action of the isolated substance (chapter I) and for the target-oriented prospection of natural products (chapter II). In the first study, chlorizidine A, isolated from Streptomyces sp. and with known antitumor potential, was subjected to the immunoaffinity fluorescence (IAF) approach to elucidate its molecular target and to investigate its cytotoxic activity in tumor cells. The IAF probe of chlorizidine A was synthesized from the coupling of the IAF tag to the natural molecule and, in cell colocalization studies, the probe was initially dispersed in the cytosol and subsequently concentrated in the lysosomes of HCT-116 cells. Enolase 1 (ENO1) and GAPDH, two proteins of the glycolytic pathway, were detected in higher abundance in the treated cells. Chlorizidine A and its IAF probe were able to bind to recombinant ENO1, indicating that as the target protein of the natural molecule, while evaluation of the cytotoxic activity suggested the occurrence of inhibition of cell proliferation related to changes occurring in the G0/G1 phase, by inhibition of the glycolytic pathway. The second study used a bioaffinity-based assay to identify substances with anticancer potential that bind to inhibitors of apoptosis proteins (IAPs) in extracts of marine actinobacteria. The bioaffinity assay using the recombinant target proteins survivin, livin and BIR3-XIAP, and 21 extracts of marine actinobacteria, recovered 100 hits, most of which were detected for the three target proteins. The molecular network with 21 analyzed extracts allowed the identification of groups of compounds corresponding to seven different classes of known natural products. Dereplication of the 12 hits identified for strain BRA177 (Actinomadura sp.) through the DNP and AntiMarin databases, along with comparison of the respective fragmentation spectra, indicated that these compounds belong to the classes of tambjamines and prodiginines. The chemical fractionation of the BRA177 extract led to the isolation of three prodiginines, methylcyclooctylprodigiosine (1), cyclononylprodigiosine (2) and nonylprodigiosine (3), corresponding to the hits obtained in the bioaffinity assay. Hits 2 and 3 were cytotoxic to tumor cell lines by short- and long-term exposure. Thus, these findings open perspectives for investigation of the relationship between the inhibition of glycolytic pathway and the anticancer activity of chlorizidine A, and for investigation of the BRA117’s prodiginines and tambjamines interaction with IAPs proteins.