Table 1.
Screening of the compounds using FBQSAR.
Tanimoto combo scores of the top five compounds and their predicted pIC50 values.
Fig 1.
Screening of the compounds using FBQSAR.
The scatter plot shows the predicted pIC50 values of the training and test set of the compounds used during the QSAR analysis. The top five compounds with an higher pIC50 were selected for further analysis.
Fig 2.
Insilico screening of the compounds.
This figure depicts the procedure used during the screening of the compounds.
Table 2.
IC50 prediction of the compounds in L.major promastigotes.
IC50 values of compounds post 48h of treatment. Compounds giving less than 50% inhibition have been reported to have IC50 >1000 μM.
Fig 3.
Characterisitics of the nanoliposomal formulation.
a) Determination of the hydrodynamic diameter of the nanoparticles using DLS. Particles are sub 200nm in size b) Shape of the nanoparticles as observed by FESEM. Nanoparticles appear monodispersive in nature and are spherical in nature c) AFM characterization of the nanoparticles d) Release profile of compound 2 from the nanoliposomes. The nanoparticles ensure that around 93% drug release occurs over a period of 50h. The designed nanoparticles are capable of releasing the drug in a slow and sustained manner.
Fig 4.
Testing the efficacy and cytotoxicity of the nanoliposomal formulation.
a) IC50 prediction of C2NL. L.major promastigotes were treated for 48hours with 30μM of the C2 containing nanoliposomes. Bar graph indicates the percentage viability of parasites. A comparison has also been made with that of empty liposomes. Inhibitory effect was evaluated using MTT assay. b) Cytotoxicity of the nanoliposomes over J774 macrophage cell line. c) Microscopic analysis of the treated parasites. Parasites treated with C2NL show abnormal morphology and reduced motility. d) SEM images of the treated and untreated promastigotes. Treated promastigotes tend to round up and shrink in size. They also loose their flagella and become less motile. Bar = 1μm.
Fig 5.
C2 induces mitochondrial mediated apoptosis in L.major promastigotes.
a) FACS analysis showing the reduction of mitochondrial potential and the induction of apoptosis in the C2NL treated L.major promastigotes. Promastigotes were treated with 30μM of C2NL for a period of 48hours. The image shown is a representative image of three independent experiments b) Percentage of cells showing an increase in the number of cells in the late apoptotic phase in the treated samples as well reduction in the number of live cells. Experiment was performed in triplicates. Significance is indicated in the figures as *p<0.01. Error bars represent the standard deviation of the mean.
Fig 6.
Course of L.major infection in mice treated with C2.
a) Footpad swelling progression during drug treatment. Weekly recordings of footpad swelling are shown for the untreated and the mice treated with compound 2. Footpad swelling is expressed as the increase of the infected over the noninfected footpad. Data shown summarizes the (Mean±SEM) from 4 animals each. *p <0.05, **p<0.01. Photographs of the footpad at the end of the treatment indicate the progression of the ulceration. b) Footpad swelling progression of the miltefosine treated mice. c) Footpad swelling progression of the compound 3 treated mice d) Parasite load assay of the treated groups. Significance is indicated in the figure as **p<0.01. Every dot indicates one mice.