Leishmanicidal Activity of Isoselenocyanate Derivatives

Abstract

Conventional chemotherapy against leishmaniasis includes agents exhibiting considerable toxicity. In addition, reports of drug resistance are not uncommon. Thus, safe and effective therapies are urgently needed. Isoselenocyanate compounds have recently been identified with potential antitumor activity. It is well known that some antitumor agents demonstrate effects against Leishmania In this study, the in vitro leishmanicidal activities of several organo-selenium and organo-sulfur compounds were tested against Leishmania major and Leishmania amazonensis parasites, using promastigotes and intracellular amastigote forms. The cytotoxicity of these agents was measured in murine peritoneal macrophages and their selectivity indexes were calculated. One of the tested compounds, the isoselenocyanate derivative NISC-6, showed selectivity indexes 2- and 10-fold higher than those of the reference drug amphotericin B when evaluated in L. amazonensis and L. major, respectively. The American strain (L. amazonensis) was less sensitive to NISC-6 than L. major, showing a trend similar to that observed previously for amphotericin B. In addition, we also observed that NISC-6 significantly reduced the number of amastigotes per infected macrophage. On the other hand, we showed that NISC-6 decreases expression levels of Leishmania genes involved in the cell cycle, such as topoisomerase-2 (TOP-2), PCNA, and MCM4, therefore contributing to its leishmanicidal activity. The effect of this compound on cell cycle progression was confirmed by flow cytometry. We observed a significant increase of cells in the G₁ phase and a dramatic reduction of cells in the S phase compared to untreated cells. Altogether, our data suggest that the isoselenocyanate NISC-6 may be a promising candidate for new drug development against leishmaniasis.

Keywords: Leishmania; MCM4; PCNA; isoselenocyanate; topoisomerases.

FIG 1

Structures of sulfur and selenium compounds.

FIG 2

Dose-response curves of L. major and L. amazonensis promastigotes treated during 72 h at 26°C with the selected compounds ISC-4 (A), NISC-6 (B), NTU-2 (C) and with the reference drug amphotericin B (Ampho B) (D). Experiments were performed in triplicate. The plots show the means ± the SD of growth inhibition values measured at each concentration.

FIG 3

Effects of NISC-6 and amphotericin B (Ampho B) on intracellular amastigotes of L. major (A) and L. amazonensis (B). The drug treatments were performed at different concentrations (10 nM, 100 nM, and 1 μM NISC-6 and 1 μM amphotericin B). Bars represent the number of parasites per infected cell with L. major and L. amazonensis after 72 h of treatment. The controls represent the untreated cells. The results show the means from three independent duplicate experiments ± the SD. Significant reductions in the number of amastigotes per infected macrophages were observed (*, P < 0.05; **, P < 0.01; ***, P < 0,001).

FIG 4

Effect of NISC-6 on Leishmania replication. mRNA levels of Leishmania topoisomerase-2 (A), PCNA (C), and MCM4 (D) after 24 h of treatment without or with two concentrations of the compound NISC-6 (0, 25, and 50 nM). (B) Cleavage complex formation, measured as disintegrations per minute (dpm), after 36 h of [methyl-³H]thymidine L. major radiolabeled, followed by 3.5 h of NISC-6 (50 nM), etoposide (20 μM) or no treatment (no drug exposure). The bars represent the means ± the SD from two independent experiments performed in sextuplicate. Significant reduction in the levels of Leishmania TOP-2, PCNA, and MCM4 after NISC-6 treatments was observed (*, P < 0.05).

FIG 5

Effect of NISC-6 on Leishmania cell cycle progression. (A) Percent of cells at the different cell cycle phases (G₁, S, and G₂) after 24 h of treatment without or with two concentrations of the compound NISC-6 (0, 25, and 50 nM). The bars represent the means ± the SD from two independent experiments performed in duplicate. (B) Representative histogram plot of DNA content against cell numbers. A significant increase in the percentage of cells in the G₁ phase and significant reduction in the percentage of cells in the S phase after NISC-6 treatments was observed (*, P < 0.05).