Mechanism of Action of Miltefosine on Leishmania donovani Involves the Impairment of Acidocalcisome Function and the Activation of the Sphingosine-Dependent Plasma Membrane Ca ²⁺ Channel

Abstract

<i>Leishmania donovani</i> 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 <i>L. donovani</i> 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 <i>c</i> 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 Ca²⁺ homeostasis represents an important object for the action of drugs in trypanosomatids. Recently, we have described a plasma membrane Ca²⁺ channel in <i>Leishmania mexicana</i>, which is similar to the L-type voltage-gated Ca²⁺ channel (VGCC) present in humans. Remarkably, the parasite Ca²⁺ 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 Ca²⁺ channel from <i>L. donovani</i> Interestingly, nifedipine, the classical antagonist of the human channel, was not able to fully block the parasite plasma membrane Ca²⁺ 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 <i>L. donovani</i>, inducing the rapid alkalinization of these important organelles. In conclusion, we demonstrate two new mechanisms of action of miltefosine in <i>L. donovani</i>, both related to disruption of parasite Ca²⁺ homeostasis.