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.