Article Figures & Data
Figures
- FIG 1
(A) Ribbon diagrams of the Ld-APX enzyme generated via homology-based modeling (red) with the predicted active site residues (in stick form and blue) making the binding pocket using PyMOL. (B) Superimposed structure of the target and its corresponding template enzyme.
- FIG 2
(A and B) Quality of the model as analyzed via Ramachandran plot (A), showing that most of the residues are in the allowed regions (B). (C) ProSA Web server image showing that the generated model is reliable.
- FIG 3
Postdocking analysis of docked complexes made between the Ld-APX enzyme and the top four compounds. (A) The Ld-APX enzyme is depicted as a cartoon model with brown color and the top screened inhibitor having ID ZINC96021026 is presented as a stick model in red color. (B) Second ranked inhibitor with ID ZINC43763954 is shown in green color inside the cavity of the modeled protein. (C) Inhibitor ZINC43061727 is depicted in blue color and (D) inhibitor ZINC14951505 is shown in purple color inside the cavity of Ld-APX enzyme. Ligplot was used for the representation of the interactions made by the different ligands with the Ld-APX protein.
- FIG 4
Molecular dynamics simulation analysis during a time span of 40 ns. (A) RMSD plot as a function of time. (B) RMSF of backbone atoms of the apo Ld-APX enzyme and all the docked complexes. (C) Rg values of all the systems. (D) The average distances between the Ld-APX protein and all four inhibitors are plotted against time. Please refer to the supplemental material for color interpretation.
- FIG 5
(A) Viability of promastigotes in the presence of various concentrations of ML-240 for an 18-h XTT assay. (B) Photomicrographs of leishmanicidal effects of increasing concentrations of miltefosine or ML-240. (C and D) Assessment of the percentage of infected macrophages and amastigotes/100 macrophages in the presence of increasing concentrations of (C) miltefosine or (D) ML-240 following coculture for 2 days. (E) Measurements of EC50 and EC90 of ML-240 against the promastigotes of L. donovani for extended periods of time. (F) Measurements of EC50 for miltefosine or ML-240 against promastigotes and amastigotes and toxicity against RAW 264.7 cells. Data are presented as the mean ± SD; n = 5.
- FIG 6
(A to G) TUNEL staining of promastigotes in the presence of increasing concentrations of miltefosine or ML-240. (H) Quantification of TUNEL-positive cells in the presence of the indicated treatment. (I) Degradation pattern of genomic DNA of promastigotes in the presence of the indicated treatment. Data are presented as the mean ± SD; n = 3.
- FIG 7
Assessment of mitochondrial ROS generation in promastigotes following the indicated treatment. (A) Untreated; (B) miltefosine (10 μ); (C) ML-240 (10 μ); (D) ML-240 (25 μM) (magnification ×1,000). ML-240 triggers ROS production and increases mitochondrial oxidative stress in L. donovani promastigotes. Following exposure to ML-240 or miltefosine, the cells were stained with CellROX Green and fixed to measure the oxidative stress level. Green signals indicate the presence of oxidative stress in promastigotes. (iii) The percentage of CellROX-positive cells was quantified on 10 random fields per treatment group. (ii) Cells were stained with MitoTracker Red CMXRos to detect mitochondrial ROS (red). (E and F) Flow cytometric determination of intracellular ROS generation in L. donovani promastigotes following treatment with (E) miltefosine or (F) ML-240 using CellROX Green reagent. (G and H) Time-dependent quantization of ROS generation in the presence of various concentrations of (G) miltefosine or (H) ML-240 using CellROX Green. Data are presented as the mean ± SD; n = 5.
- FIG 8
(A) Spectrophotometric determination of ascorbate peroxidase activity in the presence of increasing concentrations of ML-240. (B) Comparative analysis of ascorbate peroxidase activity in promastigotes following treatment with miltefosine or ML-240. (C to E) Inhibition mechanism and binding mode of ML-240. The initial enzyme activity of APX was measured as a function of the concentration of H2O2 at a fixed concentration of inhibitors. ML-240 acts in a noncompetitive manner as shown in the Lineweaver-Burk plot. (F) Measurement of intracellular H2O2 production in promastigotes by a luminescence-based assay in the presence of increasing concentrations of ML-240. Data are presented as the mean ± SD; n = 5.
- FIG 9
Antileishmanial efficacy of ML-240 in established L. donovani infection. Three-week-postinfected BALB/c mice were left either untreated (infection control) or treated intraperitoneally with ML-240 (5 or 10 mg/kg body weight) or miltefosine (5 mg/kg body weight) on alternate days for 10 days as described in Materials and Methods. (A) Phenotypic illustration of hepatosplenomegaly in L. donovani-infected BALB/c mice with enlarged abdomen. (B) Body weights of L. donovani-infected animals compared to treated groups. (C and D) Enlargement of (C) spleen and (D) liver in L. donovani-infected BALB/c mice compared to the control group 10 days posttreatment. (E and F) Weight of (E) spleen and (F) liver in the infected control compared to treated groups. (G and I) Splenic and (H and J) hepatic parasite burdens (LDU) were determined by counting the parasites in stamp smears (magnification, ×1,000). (K) Histopathological alterations in the paraffin section and hematoxylin and eosin (H&E)-stained section in the liver of L. donovani-infected BALB/c mice after treatment with either miltefosine or ML-240. Microscopic observation of inflammatory infiltrates in the periportal areas of liver sections of (i) noninfected animals, (ii) the untreated infected group, (iii) infected animals treated with 5.0 mg/kg/day of miltefosine, and infected animals treated with (iv) 5 mg/kg/day and (v) 10 mg/kg/day of ML-240. Magnification, ×400. Data are presented as the mean ± SE for five animals per group. Data were tested with ANOVA. Differences between the means were assessed for statistical significance with Tukey’s test (**, P ≤ 0.01; ***, P ≤ 0.001). Results are from one of three representative experiments.
Tables
Supplemental material
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Supplemental material
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