Fig 1.
Chemical structure of 2HF used in this study.
Fig 2.
2HF effects against wild-type or antimony-resistant promastigotes.
Wild-type or antimony-resistant L. amazonensis promastigotes were incubated in Schneider’s Drosophila medium in the absence or presence of increasing concentrations of 2HF (3–96 μM) for 24 hours. The number of parasites was determined by direct counting using a Neubauer chamber. In the control (absence of 2HF), the same volume of DMSO (0.2% v/v; solvent of 2HF) was added to the growth medium. The values are presented as the mean ± standard error of three different experiments. a) Wild-type (5 passages), b) Antimony-resistant (32 passages), c) Wild-type comparative (32 passages). The IC50 was calculated via nonlinear regression using GraphPad Prism 6.0. * indicates significant difference relative to control (p < 0.05).
Table 1.
Comparative IC50 for 2HF against wild-type and antimony-resistant L. amazonensis promastigote.
Fig 3.
Effect of 2HF and meglumine antimoniate on L. amazonensis-infected macrophages.
Macrophages were infected with wild-type or antimony-resistant L. amazonensis promastigotes at 37°C and 5% CO2. After 3 hours of infection, the remaining promastigotes were removed. After 18 hours, the infected macrophages were incubated in the absence or presence of increasing concentrations of 2HF (3–48 μM) or meglumine antimoniate (3.125–200 μM) for 72 hours. The infection index was determined using light microscopy. At least 200 macrophages were counted on each coverslip in duplicate. The values shown represent the mean ± standard error of three independent experiments. In the control samples (absence of 2HF), a similar volume of vehicle (0.2% DMSO) was added to the cells. Panel A and B: Wild-type and antimony-resistant cells, respectively, treated with meglumine antimoniate; Panel C and D: Wild-type and antimony-resistant, respectively, treated with 2HF. The values are presented as the mean ± standard error of three different experiments. 2HF: 2’-hydroxyflavanone; WT: Wild-type; R: Antimony-resistant; Vehicle: RPMI-1640 medium with 0.2% DMSO. * indicates significant difference relative to control (p < 0.05).
Fig 4.
Illustrative photos of 2HF and meglumine antimoniate against L. amazonensis-infected macrophages.
Macrophages infected with wild-type L. amazonensis promastigotes (Panel A) or antimony-resistant L. amazonensis promastigotes (Panel B). Scale bars correspond to 10 μm. Black arrows indicate the presence of amastigotes. 2HF: 2’-hydroxyflavanone.
Table 2.
Comparative IC50 of meglumine antimoniate and 2HF against L. amazonensis-infected macrophages using wild-type and antimony-resistant L. amazonensis promastigotes.
Table 3.
2HF in silico ADMET predictions.
Fig 5.
In vivo effects of 2HF and meglumine antimoniate using wild-type L. amazonensis.
BALB/c mice were infected in the right ear with 2 × 106 wild-type L. amazonensis promastigotes. Panel A: Lesion development on the animals treated orally with 2HF (50 mg/kg/day), intraperitoneally with meglumine antimoniate (100 mg/kg/day) and with an oral suspension added to DMSO (0.2% v/v) (2HF vehicle). The treatment started seven days post-infection and was given once daily seven times per week until the end of the experiment (day 42). Panel B: Parasite burden of the L. amazonensis-infected BALB/c mice untreated or treated with 2HF (50 mg/kg/day) or meglumine antimoniate (100 mg/kg/day). Ear parasite loads were determined via a limiting dilution assay. Data are expressed as the means ± standard errors. These data represent two independent experiments with five mice per group each (n = 5). *, ** and *** indicate significant differences relative to the control group and #, ##, ### indicate significant differences relative to 2HF (p < 0.05; p< 0.01 and p < 0.001, respectively); Panel C: Illustrative lesion photos of a representative infected ear treated with the vehicle (left photo), 2HF (center photo) and meglumine antimoniate (right photo). 2HF = 2’-Hydroxyflavanone.
Fig 6.
Leishmanicidal effect of 2HF and meglumine antimoniate in antimony-resistant L. amazonensis -infected BALB/c mice.
BALB/c mice were infected in the right ear with 4 × 106 antimony-resistant L. amazonensis promastigotes. Panel A: Lesion development on the animals treated orally with 2HF (50 mg/kg/day). Panel B: Lesion development on the animals treated intraperitoneally with meglumine antimoniate (100 mg/kg/day). The untreated mice (control group) were treated with an oral suspension added to DMSO (0.2% v/v) (2HF vehicle). The treatment started seven days post-infection and was given once daily seven times per week until the end of the experiment (day 42). Panel C: Parasite burden of the L. amazonensis-infected BALB/c mice untreated or treated with 2HF (50 mg/kg/day) or meglumine antimoniate (100 mg/kg/day). Ear parasite loads were determined via a limiting dilution assay. Data are expressed as the means ± standard errors. These data represent one independent experiment with five mice per group each (n = 5). *, ** and *** indicate significant differences relative to the control group (p < 0.05; p < 0.01 and p < 0.001, respectively) and ## indicate significant differences relative to 2HF (p < 0.01); 2HF = 2’-Hydroxyflavanone; ns = No statistical significance.
Fig 7.
Resistance confirmation in in vivo recovered promastigotes.
L. amazonensis promastigotes were recovered from the in vivo limiting dilution experiment from each treated group and cultivated with Schneider’s Drosophila medium. Promastigotes were incubated in the presence or absence of the potassium antimony tartrate (SbIII) (0.3–5000 μM) for 72 hours. The viability was measured by resazurin. The IC50 for resistance confirmation was calculated via nonlinear regression using GraphPad Prism 6.0. The values are presented as the mean ± standard error of two different experiments. Panel A: Wild-type promastigotes; Panel B: Promastigotes recovered from the vehicle treatment group; Panel C: Promastigotes recovered from the 2HF-treated group; Panel D: Promastigotes recovered from the meglumine antimoniate-treated group.
Table 4.
Comparative IC50 for antimonial against in vivo recovered promastigotes.