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Leishmaniasis is parasitic disease that is an important problem of public health worldwide. Intramuscularly administered glucantime and pentostam are the most common drugs used for treatment of this disease, but they have significant limitations due to toxicity and increasing resistance. A recent breakthrough has been the introduction of orally administered miltefosine for the treatment of visceral, cutaneous, and mucocutaneous leishmaniasis, but the relative high cost and concerns about teratogenicity have limited the use of this drug. Searching for alternative drugs, we previously demonstrated that the antiarrhythmic drug amiodarone is active against Leishmania mexicana promastigotes and intracellular amastigotes, acting via disruption of intracellular Ca 2؉ homeostasis (specifically at the mitochondrion and the acidocalcisomes of these parasites) and through inhibition of the parasite's de novo sterol biosynthesis (X. Serrano-Martín, Y. García-Marchan, A. Fernandez, N. Rodriguez, H. Rojas, G. Visbal, and G. Benaim, Antimicrob. Agents Chemother. 53:1403-1410, 2009). In the present work, we found that miltefosine also disrupts the parasite's intracellular Ca 2؉ homeostasis, in this case by inducing a large increase in intracellular Ca 2؉ levels, probably through the activation of a plasma membrane Ca 2؉ channel. We also investigated the in vitro and in vivo activities of amiodarone and miltefosine, used alone or in combination, on L. mexicana. It was found that the drug combination had synergistic effects on the proliferation of intracellular amastigotes growing inside macrophages and led 90% of parasitological cures in a murine model of leishmaniasis, as revealed by a PCR assay using a novel DNA sequence specific for L. mexicana.

Cystathionine β-synthase (CBS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)), found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.

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