Fig 1.
The efficacy of Miransertib (ARQ092) as compared to miltefosine on promastigote forms of Leishmania amazonensis (A) and Leishmania donovani (B) was measured in MTT assays after 24 h treatment of each drug at increasing concentrations. The proportion of surviving parasites was analyzed by GraphPad prism and the EC50 was calculated from those plots. The data was compiled from at least 3 independent experiments.
Fig 2.
Determination of anti-leishmanial potential of Miransertib (ARQ092) on Leishmania in infected macrophages.
(A) dTHP-1 cells (human macrophages) were infected with L. donovani promastigotes for 24 hours and then treated with indicated concentrations of Miransertib for an additional 24 hours. At the end of the experiment, infected cells with and without Miransertib treatment were washed, and internalized parasites were released by lysing the cells using a mild concentration of SDS, followed by transfer to transformation medium as described in “Materials and Methods”. Transformed motile promastigotes were counted. The data was compiled from three independent experiments done in duplicates. B) RAW264.7 macrophages were infected with L. amazonensis promastigotes. After 24 hours, infections cells were treated with indicated concentrations of ARQ092 or miltefosine and incubated for an additional 24 hrs. Cells on coverslips were Giemsa-stained and enumerated. The proportion of infected cells was compiled and plotted in GraphPad. Data were compiled from at least 3 experiments.
Fig 3.
Effect of Miransertib on the parasitic burden in L. donovani-infected BALB/c mice.
Female BALB/c mice (n = 4) were infected intravenously with approximately 107 stationary phase L. donovani promastigotes on day -7. On day 0, treatment was begun with Miransertib (50 or 75 mg/kg) orally or 20 mg/kg of miltefosine orally. The control group received only vehicle orally. Drugs or vehicle were given five days a week for 4 weeks. The parasite survival in spleens (A) and livers (B) were determined at the end of the experiment as described in “Materials and Methods”. Significance of differences between non-treated and treated was tested by student t-test *p<0.05; **p<0.01 and***p<0.001.
Fig 4.
Miransertib partially controls L. amazonensis infection.
Groups of 5 BALB/c mice were administered saline or the drug concentrations indicated orally for 5 days a week. Kinetics of infection was determined by weekly measurement of lesions in infected foot compared to the contralateral foot. Each point is the average ± SE **: p<0.005 ***: p<0.0005.
Fig 5.
Leishmania induced autophagy is enhanced in the presence of Miransertib by inhibiting pAkt.
dTHP-1 cells were incubated with L. donovani promastigotes for 24 h. After 24 h infection, cells were treated with indicated concentrations of Miransertib for an additional 24 h (panel B). In parallel, non-infected cells were treated with Miransertib alone for the comparison (panel A), and infected cells without Miransertib were used as controls. At the end of the experiment, cells were then washed with HBSS and whole cell lysates were collected and analyzed by immunoblotting for LC3-II, pAkt and Akt (levels (panel B). Actin levels were also analyzed as loading controls. The histograms shown are densitometric analyses of relative LC3-II levels and pAkt in three independent experiments. Data are presented as mean ± SD. Densitometric analysis was performed in ImageJ. The results of LC3-II are expressed normalized to actin where as the results of pAkt are expressed normalized to Akt.
Fig 6.
Miransertib treatment enhances the level of macrophage autophagy in Leishmania infected mice spleens.
Macrophages from Leishmania-infected mice spleens, and Leishmania-infected and treated with Miransertib or miltefosine mice spleens were isolated as described in “Materials and Methods” and analysed for the levels of LC3-II. The histogram shows the levels of LC3-II in macrophages of Miransertib-treated or Miltefosine-treated leishmania infected spleens normalized to LC3-II levels in non-treated leishmania infected spleens. Data are presented as mean ± SD of macrophages obtained from spleens of 4 mice in each group. Densitometric analysis was performed in ImageJ. The results of LC3-II are expressed normalized to actin.