Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC₅₀s for nifurtimox, benznidazole, H₂O₂ and NO^. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease.

Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC₅₀ values in the range of 4.6–10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC₅₀ of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC₅₀ = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease. Polyamines are polycationic compounds essential for the regulation of cell growth and differentiation. In contrast with other protozoa, Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for polyamines; therefore the intracellular availability of these molecules depends exclusively on transport processes. It was previously demonstrated that the lack of polyamines in T. cruzi leads to its death, making the polyamine transporter an excellent therapeutic target for Chagas disease. In this work, the polyamine permease TcPAT12 was selected as a target for drug screening using 3000 FDA-approved compounds and computational simulation techniques. Using two combined virtual screening methods, isotretinoin, a well-known and safe drug used for acne treatment, bound to substrate recognition residues of TcPAT12 and was chosen for further in vitro studies. Isotretinoin inhibited not only the polyamine transport but also all tested amino acid transporters from the same protein family as TcPAT12. Interestingly, isotretinoin showed a high trypanocidal effect on trypomastigotes, with an IC₅₀ in the nanomolar range. Autophagy and apoptosis were proposed as mechanisms of parasites death induced by isotretinoin. These results suggest that isotretinoin is a promising trypanocidal drug, being a multi-target inhibitor of essential metabolites transporters.

Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the possibility of using these molecules as a route of entry of therapeutic drugs.

During three decades, only about 20 new drugs have been developed for malaria, tuberculosis and all neglected tropical diseases (NTDs). This critical situation was reached because NTDs represent only 10% of health research investments; however, they comprise about 90% of the global disease burden. Computational simulations applied in virtual screening (VS) strategies are very efficient tools to identify pharmacologically active compounds or new indications for drugs already administered for other diseases. One of the advantages of this approach is the low time-consuming and low-budget first stage, which filters for testing experimentally a group of candidate compounds with high chances of binding to the target and present trypanocidal activity. In this work, we review the most common VS strategies that have been used for the identification of new drugs with special emphasis on those applied to trypanosomiasis and leishmaniasis. Computational simulations based on the selected protein targets or their ligands are explained, including the method selection criteria, examples of successful VS campaigns applied to NTDs, a list of validated molecular targets for drug development and repositioned drugs for trypanosomatid-caused diseases. Thereby, here we present the state-of-the-art of VS and drug repurposing to conclude pointing out the future perspectives in the field.