Table 1.
The 50% inhibitory concentration (IC50) of the compounds, their selectivity against the promastigotes/amastigotes of Leishmania donovani and their haemolytic profile (HC50).
Fig 1.
Growth kinetics, cytocidal and cytostatic profile of the MMV compounds on Leishmania donovani parasites.
The growth kinetics curves of (A) MMV676600 (C03B), MMV676602 (H02B) and MMV676162 (C06D), (B) MMV688798 (H05A), MMV652003 (A03B) and MMV676604 (B03B), (E) MMV690027 (C02B), MMV688797 (E05A) and MMV688958 (G05A), (F) MMV676159 (A07E), MMV688360 (H03A) and MMV099637 (H04A), (I) MMV688793 (D05A), MMV688514 (E02A) and MMV687762 (D02A), (J) MMV688943 (C06A), MMV688796 (A06A) and MMV690028 (C05A) against Leishmania donovani promastigotes cells counted within 5 days of incubation. A concentration of 1μg/ml of the MMV compounds were used. ANOVA was used to analyze the significant differences between treatments at P<0.05. The growth reversibility profile of (C) MMV676600 (C03B), MMV676602 (H02B) and MMV676162 (C06D), (D) MMV688798 (H05A), MMV652003 (A03B) and MMV676604 (B03B), (G) MMV690027 (C02B), MMV688797 (E05A) and MMV688958 (G05A), (H) MMV676159 (A07E), MMV688360 (H03A) and MMV099637 (H04A), (K) MMV688793 (D05A), MMV688514 (E02A) and MMV687762 (D02A), (L) MMV688943 (C06A), MMV688796 (A06A) and MMV690028 (C05A) after drug withdrawal and resuspension of the cells in fresh complete media and cells counted after four days incubation. Data shows the mean of three independent experiments. UP = Untreated Parasites (Negative control) and AMPHO = amphotericin B (positive control).
Fig 2.
The effects of MMV compounds on ROS generation in amastigotes and promastigote of Leishmania donovani.
(A) Pro-oxidative and anti-oxidative profiles of MMV compound treated amastigotes shown as an index of the fluorescence intensity unit of the H2DCFDA rich dye (B and C) Pro-oxidative and anti-oxidative profile of MMV compound treated promastigotes shown as an index of the fluorescence intensity unit of the H2DCFDA rich dye.
Fig 3.
Effect of MMV688793 (D05A) and MMV688943 (C06A) compounds on Leishmania donovani promastigotes programmed cell death.
(A) Untreated cells (Negative control) and cells treated with (B) MMV688793 (D05A) (2x IC50). (C) MMV688943 (C06A) (2x IC50). (D) Percentage cell quantification of parasites in the respective stages. Statistical significance was determined using 2-way ANOVA by comparing the treated and the control. Significance was set at P<0.05. These are representative profiles of three independent experiments.
Fig 4.
Cell cycle progression of Leishmania donovani promastigotes.
Flow cytometry histograms of (A) Untreated (B) MMV688943 (C06A) (C) MMV688793 (D05A). Promastigotes were incubated with the compounds at 2x IC50 for 24 hrs. Untreated promastigotes were used as the negative control. (D) Cell quantification percentile of each cell cycle phase. The Student’s t test was used to determine statistical significance at P<0.05. All data shown are representative of three independent experiments.
Fig 5.
The effects of compounds on the promastigote nuclear / kinetoplast DNA and Mitochondria morphology.
(A) MMV688793 (D05A) (B) MMV688943 (C06A) (C) MMV688514 (E02A) (D) MMV690028 (C05A) (E) MMV688796 (A06A) (F) MMV687762 (D02A). Promastigotes were treated with the MMV compound for 24 hrs, 48hrs and 72 hrs. They were stained with DAPI and MitoTracker Red, then examined by phase contrast microscopy and fluorescence microscopy at x1000. Key: M = Normal Mitochondrion; dM = Degenerated Mitochondrion; N = Nucleus; dN = degenerated nucleus; K = Kinetoplast; fN = fragmented Nuclei DNA.