Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by <i>Leishmania (Viannia) braziliensis</i>

<div><p>Background</p><p>Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces <i>L. (L.) amazonensis</i> infection <i>in vitro</i>. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with <i>L. (V.) braziliensis</i>.</p><p>Methodology/Principal findings</p><p>Exposure of axenic <i>L. (V.) braziliensis</i> promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to <i>L. (V.) braziliensis</i>-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O⁻²) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated <i>in vivo</i>, employing BALB/c mice infected with <i>L. braziliensis</i>. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability.</p><p>Conclusion/Significance</p><p>17-AAG effectively inhibited the growth of <i>L. braziliensis</i>, both <i>in vitro</i> and <i>in vivo</i>. Given the chronicity of <i>L. (V.) braziliensis</i> infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis.</p></div

17-AAG induces killing of <i>Leishmania (V.) braziliensis</i> promastigotes in a dose-dependent and irreversible manner.

<p><i>L. (V.) braziliensis</i> promastigotes were exposed to increasing concentrations of 17-AAG, to vehicle alone (DMSO) or were left unexposed (Lb) for 48 h. (A) The number of viable parasites was evaluated by direct counting. <i>L.(V) braziliensis</i> promastigotes were treated with 65 nM (IC₅₀) of 17-AAG for 24 h (B) and (C) 48 h. After washing, promastigotes were cultured for additional 48 h and the number of viable parasites was evaluated. Data, shown as mean ±SEM, are from one of two independent repeats (**<i>p</i><0.01 and ***<i>p</i><0.001).</p

<i>In vivo</i> treatment with 17-AAG decreases <i>L. (V.) braziliensis</i> infection.

<p>Mice were infected with <i>L. (V.) braziliensis</i> and four weeks later, mice were treated with 17-AAG, 3x a week for 3 weeks (boxed area) or with vehicle (DMSO) alone. (A) The course of lesion development was monitored weekly. (B) Disease burden [shown as Area Under the Curves (AUC) depicted in (A)] in mice treated with 17-AAG or injected with DMSO. Parasite load was determined at the infection site (C) and at the dLN (D), 6 weeks later, by limiting dilution analysis. Data, shown as mean ±SEM, are from one of two independent repeats, each performed with 10 mice in each group (**<i>p</i><0.01; *<i>p</i><0.05).</p

Comparison of the effects of 17-AAG and amphotericin B on the growth of intracellular <i>L. (V.) braziliensis</i>.

<p><i>L. (V.) braziliensis-</i>infected macrophages were treated with 17-AAG or with amphotericin B (AMB) for 48 h. The number of viable parasites was evaluated by further culture in Schneider medium, free of 17-AAG. Data, shown as mean ±SEM, are from one of two independent repeats (***<i>p</i><0.001). (ND, not detected).</p

Treatment with 17-AAG controls <i>L. (V.) braziliensis</i> replication inside macrophages.

<p><i>L. (V.) braziliensis-</i> infected macrophages were treated with increasing concentrations of 17-AAG or with vehicle alone (DMSO). After 12–72 h, glass coverslips were stained with H&E and assessed for the percentage of infected macrophages (A) and the number of amastigotes per 100 macrophages (B) by light microscopy. Data, shown as mean ±SEM, are shown as the percentage of DMSO -treated cultures, from one of three independent repeats (*<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001).</p

Treatment with 17-AAG reduces intracellular <i>L. (V.) braziliensis</i> viability.

<p><i>L. (V.) braziliensis</i>- infected macrophages were treated for 24 h with increasing concentrations of 17-AAG or with vehicle alone (DMSO). The number of viable parasites was evaluated by further culture (5 days) in Schneider medium, free of 17-AAG (A). Infected macrophages were treated with 220 nM (IC₅₀) of 17-AAG for 24–72 h. The number of viable parasites was evaluated by further culture for five days in Schneider medium, free of 17-AAG (B). Data, shown as mean ±SEM, are from one of two independent repeats (**<i>p</i><0.01 and *** <i>p</i><0.001).</p

Treatment with 17-AAG down regulates ROS and cytokine production in <i>L. (V.) braziliensis-infected</i> cells.

<p><i>L. (V.) braziliensis-</i>infected macrophages were treated with 17-AAG (220nM) + IFN-. After 48h, supernatants were assayed for nitrite production (A) and for presence of (B) superoxide, following addition of hydroxylamine. The presence of secreted (C) TNF-α, (D) IL-6 and (E) CCL2 was determined in culture supernatants by Cytometric Bead Array, after 24 h of treatment. Data, shown as mean ± SEM, are from one of two independent repeats (**<i>p</i><0.01; *<i>p</i><0.05).</p