is the causing agent of visceral leishmaniasis, a common infection that affects millions of people from the most underdeveloped countries. Miltefosine is the only oral drug to treat infections caused by Nevertheless, its mechanism of action is not well understood. While miltefosine inhibits the synthesis of phosphatidylcholine and also affects the parasite mitochondrion, inhibiting the cytochrome oxidase, it is to be expected that this potent drug also produces its effect through other targets. In this context, it has been reported that the disruption of the intracellular Ca homeostasis represents an important object for the action of drugs in trypanosomatids. Recently, we have described a plasma membrane Ca channel in , which is similar to the L-type voltage-gated Ca channel (VGCC) present in humans. Remarkably, the parasite Ca channel is activated by sphingosine, while the L-type VGCC is not affected by this sphingolipid. In the present work we demonstrated that, similarly to sphingosine, miltefosine is able to activate the plasma membrane Ca channel from Interestingly, nifedipine, the classical antagonist of the human channel, was not able to fully block the parasite plasma membrane Ca channel, indicating that the mechanism of interaction is not identical to that of sphingosine. In this work we also show that miltefosine is able to strongly affect the acidocalcisomes from , inducing the rapid alkalinization of these important organelles. In conclusion, we demonstrate two new mechanisms of action of miltefosine in, both related to disruption of parasite Ca homeostasis.

Trypanosoma cruzi is the causative agent of Chagas disease. The only two drugs accepted for the treatment of this infection are benznidazole and nifurtimox, which are of limited use in the predominant chronic phase. On the search for new drugs, the intracellular Ca2+ regulation has been postulated as a possible target, due to differences found between host cells and the parasite. The mechanisms involved in the intracellular Ca2+ regulation of T. cruzi have been partially elucidated. However, nothing is known about a putative channel responsible for the Ca2+ entry into this parasite. In contrast, in Leishmania spp., a closely related hemoflagelate, a sphingosine‐activated plasma membrane Ca2+ channel has been recently described. The latter resembles the L‐type voltage‐gated Ca2+ channel present in humans, but with distinct characteristics. This channel is one of the main targets concerning the mechanism of action of miltefosine, the unique oral drug approved against leishmaniasis. In the present work, we describe for the first time, the electrophysiological characterization of a sphingosine‐activated Ca2+ channel of T. cruzi by reconstituting plasma membrane vesicles into giant liposomes and patch clamp. This channel shares some characteristic as activation by Bay K8644 and inhibition by channel blockers such as nifedipine. However, the T. cruzi channel differs from the L‐type VGCC in its activation by sphingosine and/or miltefosine. Albeit the conductance for each, Ba2+, Ca2+ and Sr2+ was similar, the parasite channel appears not to be voltage dependent. A gene that presents homology in critical amino acids with its human ortholog Ca2+ channel was identified.

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Chagas disease is a neglected tropical affection caused by the protozoan parasite Trypanosoma cruzi. There is no current effective treatment since the only two available drugs have a limited efficacy and produce side effects. Thus, investigation efforts have been directed to the identification of new drug leads. In this context, Ca regulating mechanisms have been postulated as targets for antiparasitic compounds, since they present paramount differences when compared to host cells. Amiodarone is an antiarrhythmic with demonstrated trypanocidal activity acting through the disruption of the parasite intracellular Ca homeostasis. We now report the effect of a benzofuran derivative based on the structure of amiodarone on T. cruzi. This derivative was able to inhibit the growth of epimastigotes in culture and of amastigotes inside infected cells, the clinically relevant phase. We also show that this compound, similarly to amiodarone, disrupts Ca homeostasis in T. cruzi epimastigotes, via two organelles involved in the intracellular Ca regulation and the bioenergetics of the parasite. We demonstrate that the benzofuran derivative was able to totally collapse the membrane potential of the unique giant mitochondrion of the parasite and simultaneously produced the alkalinization of the acidocalcisomes. Both effects are evidenced by a large increase in the intracellular Ca concentration of T. cruzi.

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