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Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Upregulation of glycolysis and the pentose phosphate pathway (PPP) is a major characteristic of the metabolic reprogramming of cancer and provides cancer cells with energy and vital metabolites to support their rapid proliferation. Targeting glycolysis and the PPP has emerged as a promising antitumor therapeutic strategy. Marine natural products are attractive sources for anticancer therapeutics, as evidenced by the antitumor drug Yondelis. Mycoepoxydiene (MED) is a natural product isolated from a marine fungus that has shown promising inhibitory efficacy against HeLa cells in vitro. We used a proteomic approach with two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to explore the cellular targets of MED and to unravel the molecular mechanisms underlying the antitumor activity of MED in HeLa cells. Our proteomic data showed that triosephosphate isomerase (TPI) and 6-phosphogluconolactonase (PGLS), which participate in glycolysis and the PPP, respectively, were significantly downregulated by MED treatment. Functional studies revealed that the expression levels of several other enzymes involved in glycolysis and the PPP, including hexokinase 2 (HK2), phosphofructokinase 1 (PFKM), aldolase A (ALDOA), enolase 1 (ENO1), lactate dehydrogenase A (LDHA), and glucose-6-phosphate dehydrogenase (G6PD), were also reduced in a dose-dependent manner. Moreover, the LDHA and G6PD enzymatic activities in HeLa cells were inhibited by MED, and overexpression of these downregulated enzymes rescued HeLa cells from the growth inhibition induced by MED. Our data suggest that MED suppresses HeLa cell growth by inhibiting glycolysis and the PPP, which provides a mechanistic basis for the development of new therapeutics against cervical cancer.