Leishmanicidal Effect of Synthetic <i>trans</i>-Resveratrol Analogs

<div><p>Background</p><p>Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the <i>trans</i>-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against <i>Leishmania amazonensis</i>.</p><p>Methodology/Principal Findings</p><p>Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-<i>L</i>. <i>amazonensis</i> activity with IC₅₀ values of 18 μM, 65 μM, 95 μM and 65 μM for promastigotes, respectively. For intracellular amastigotes, the IC₅₀ values of the analogs were 33.2 μM, 45 μM, 29 μM and 30.5 μM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death.</p><p>Conclusion/Significance</p><p>Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.</p></div

<i>In silico</i> evaluations of Pterostilbene, Piceatannol, Polydatin, Oxyresveratrol and Amphotericin B.

<p>(<b>A</b>) Comparative evaluation of drug-likeness profile; (<b>B</b>) drug-score; and (<b>C</b>) toxicity profile obtained by <i>in silico</i> evaluation using Osiris^® software.</p

Effects of trans-Resveratrol on ROS production by murine peritoneal macrophages.

<p>Uninfected macrophages (10⁵) and macrophages infected with <i>Leishmania amazonensis</i> at a 10:1 ratio stimulated or not with PMA were incubated in the presence or absence of 33.2 μM pterostilbene (<b>A</b>), 45 μM piceatannol (<b>C</b>), 29 μM polydatin (<b>E</b>) or 30.5 μM oxyresveratrol (<b>G</b>) for 30 minutes. The data represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01, *** P <0.0001.</p

Effects of <i>trans</i>-Resveratrol on nitric oxide (NO) production by murine peritoneal macrophages.

<p>Uninfected macrophages (10⁵) and macrophages infected with <i>Leishmania amazonensis</i> at a 10:1 ratio stimulated with LPS or left unstimulated were incubated in the presence or absence of 33.2 μM pterostilbene (<b>A</b>), 45 μM piceatannol (<b>C</b>), 29 μM polydatin (<b>E</b>) and 30.5 μM oxyresveratrol (<b>G</b>). NO production was evaluated after 48h of treatment by the Griess method. The results of two independent experiments performed in duplicate are shown as the mean ± SEM nitrite concentration. * P <0.05, ** P <0.001, *** P<0.0001. Scavenger effect analysis was performed in a cell-free system by incubating SNAP solution (1 mM) as the NO donor with the same concentrations of <i>trans</i>-resveratrol used in NO (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>). Rutin (1 mM), an NO scavenger, was used as positive control, and RPMI medium served as negative control. Nitrite levels were determined by the Griess method. The data represent the mean ± SEM of three independent experiments. * P <0.05, ** P <0.01, *** P <0.0001.</p

Lipinski´s rule of five of <i>trans</i>-Resveratrol Analogs and Amphotericin B.

<p>ClogP—octanol/water coefficiente partition; MW—molecular weight; MV—molecular volume;</p><p>HBD—number of hydrogen bonds donated; HBA—number of hydrogen bond accepted.</p><p>Lipinski´s rule of five of <i>trans</i>-Resveratrol Analogs and Amphotericin B.</p

Analysis of <i>Leishmania amazonensis</i> promastigotes cell cycle.

<p>^# Results are the mean ± SEM of at least three independent experiments.</p><p>*** P <0.001, in relation to control.</p><p>Analysis of <i>Leishmania amazonensis</i> promastigotes cell cycle.</p

<i>In vitro</i> anti-leishmanial activity and cytotoxicity results for <i>trans</i>-Resveratrol Analogs.

<p>* The selectivity index (SI) was calculated as the ratio of CC₅₀ on murine peritoneal macrophages to IC₅₀ on <i>L</i>. <i>amazonensis</i> intracellular amastigotes.</p><p>n.d.–not determined.</p><p><i>In vitro</i> anti-leishmanial activity and cytotoxicity results for <i>trans</i>-Resveratrol Analogs.</p

Incidental death of <i>L</i>. <i>amazonensis</i> parasites treated with Piceatannol.

<p>Promastigotes were treated or not with piceatannol and the incidental death measured by the expression of Annexin V. Untreated parasites control (<b>A</b>), 1% DMSO (<b>B</b>), parasites treated with piceatannol at 100 μM (<b>C</b>), 200 μM (<b>D</b>), 400 μM (<b>E</b>) and 30 μM miltefosine (<b>F</b>) are shown as a representative result. The percentage of Annexin-V positive promastigotes, treated as above, shown as mean ± SEM of three independent experiments (<b>G,</b> bars represent Annexin⁺ plus Annexin⁺/PI⁺ cells). <i>Leishmania</i> mitochondrial activity. Promastigotes were cultured 48h in the presence or absence of 65 μM piceatannol and 1% DMSO (vehicle). The mitochondrial membrane potential (ΔΨm) was evaluated using JC-1 (<b>H</b>). Results expressed as red/green fluorescence ratios, represent the mean ± SEM of 3 independent experiments. **P<0.001, *** P <0.0001, compared to control. Milte = Miltefosine.</p

Scanning electron microscopy of <i>Oncopeltus fasciatus</i> salivary glands infected with <i>Phytomonas serpens</i>.

<p>The parasites were injected laterally into the thorax of the insects and the salivary glands were explanted at different time points after injection. (<b>A</b>) Outer surface of the salivary gland showing a high number of parasites between two salivary gland lobes and a few parasites attached to the gland, 48 h post-infection. Scale bar: 10 µm. (<b>B–C</b>) Large numbers of parasites bound to the salivary gland, 72 h post-infection. Scale bars: 100 µm (<b>B</b>) and 50 µm (<b>C</b>). SG: salivary gland; P: parasite; SGL1, SGL2 and SGL3: salivary gland lobes. The asterisk (*) indicates the salivary duct. For further details see the Methods.</p

Immunoblotting of the 130 kDa salivary gland protein probed with anti-human laminin-5 β3 chain antibodies.

<p>Total salivary gland proteins were extracted and separated by 10% SDS-PAGE (<b>a</b>). The 130 kDa band was cut and purified from the gel and the purity of the product was evaluated by 10% SDS-PAGE stained with silver nitrate (<b>b</b>). The purified 130 kDa protein was probed with anti-human laminin-5 β3 chain antibodies (<b>c</b>). The arrow shows the position of the 130 kDa protein on SDS-PAGE and the stained band by immunoblotting.</p