Rationally Design of a Proline Transporter Inhibitor with Anti-Trypanosoma Cruzi Activity

L-Proline is an important amino acid for the pathogenic protists belonging to <i>Trypanosoma</i> and <i>Leishmania </i>genera. In <i>Trypanosoma cruzi</i>, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses, including nutritional and osmotic as well as oxidative imbalance. In this study, we explore the L-Proline uptake as a chemotherapeutic target for <i>T. cruzi</i>. For this, we propose a novel rational to design inhibitors containing this amino acid as a recognizable motif. This rational consists of conjugating the amino acid (proline in this case) to a linker and a variable region able to block the transporter. We obtained a series of sixteen 1,2,3-triazolyl-proline derivatives through alkylation and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) for <i>in vitro</i> screening against <i>T. cruzi </i>epimastigotes, trypanocidal activity and proline uptake. We successfully obtained inhibitors that are able to interfere with the amino acid uptake, which validated the first example of a rationally designed chemotherapeutic agent targeting a metabolite's transport. Additionally, we designed and prepared fluorescent analogues of the inhibitors that were successfully taken up by <i>T. cruzi</i>, allowing following up their intracellular fate. In conclusion, we successfully designed and produced a series of metabolite uptake inhibitors. This is one of few examples of rationally designed amino acid transporter inhibitor, being the first case where the strategy is applied on the development of chemotherapy against Chagas disease. This unprecedented development is remarkable having in mind that only a small percent of the metabolite transporters has been studied at the structural and/or molecular level.