Fig 1.
Route of synthesis to obtain the aryl-thiazole derivatives (NJ series).
In the compounds, R was hydrogen, 4-chloro, 4-bromo or 4-nitro. For 2,4-dimethoxy-aryl thiazole (right side route) R = Cl for NJ03; R = NO2 for NJ06; R = Br for NJ08 and R = H for NJ12. For 3,4,5-trimethoxy-aryl thiazole (left side route) R = Cl for NJ04; R = NO2 for NJ05; R = Br for NJ07 and R = H for NJ11.
Fig 2.
Effect of NJ05, NJ07 or NJ05 + NJ07 on schistosomula viability.
(A and B) ATP quantitation using a luminescent assay to assess schistosomula viability under NJ series compounds exposure. Schistosomula (100-120/well) were incubated with the indicated concentrations of NJ05, NJ07, NJ05 + NJ07 or with vehicle (0.1% DMSO) for up to 5 days. Viability was expressed as % luminescence values relative to the control (DMSO). Mean ± SEM from three biological replicate experiments are shown. The two-way ANOVA test was used to calculate the statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001). For clarity purposes, we show only the highest p-value obtained from the two-way ANOVA test for each time point on all different concentrations tested. (C) Quantitation of schistosomula viability using propidium iodide staining; schistosomula were treated for two days with NJ05 + NJ07 at the different concentrations indicated. Percentage of viable schistosomula (non-stained with propidium iodide) is shown. For each condition tested, about 400 schistosomula were used, divided into four biological replicates. Mean ± SEM from four replicate experiments are shown. (D) Schistosomula treated with the indicated concentrations of NJ05 + NJ07 or with vehicle (0.1% DMSO) for 2 days were visualized by staining with propidium iodide (marker of dead cells; 572 nm emission filter microscope). For each concentration (indicated at the top), the upper panel shows a light microscopy image and the bottom panel shows the image of the same field with differential fluorescence detection of PI-positive parasites. Bar = 100 μm.
Fig 3.
In vitro effects of NJ05, NJ07 or NJ05 + NJ07 on the viability and motility of Schistosoma mansoni adult worms.
(A—C) Viability was estimated by the total amount of ATP available in the parasites, using a luminescent assay. Pairs of adult worms were treated for 2 days with NJ05, NJ07 or NJ05 + NJ07 at the different concentrations indicated or with vehicle (0.1% DMSO). Viability was expressed as percentage luminescence values relative to the control (DMSO). Mean ± SEM from three replicate experiments are shown, each with 10 worm pairs. (D—F) Percentage of relative motility of adult worms treated with different concentrations of NJ05, NJ07 or NJ05 + NJ07 and controls (0.1% DMSO) at different times of exposure from 1 to 3 days. Mean ± SEM of three experiments, each with 10 worm pairs. *p < 0.05, **p < 0.01 and *** p < 0.001 compared with controls. For clarity purposes, we show only the highest p-value obtained from the two-way ANOVA test for each time point on all different concentrations tested.
Fig 4.
In vitro effects of NJ05, NJ07 or NJ05 + NJ07 on pairing and oviposition of Schistosoma mansoni adult worms.
(A-C) We monitored the pairing of control couples and of couples treated with different concentrations of NJ05, NJ07 or NJ05 + NJ07 at different times of exposure from 1 to 3 days. Mean ± SEM of three experiments are shown, each with 10 worm pairs. (D-F) Number of eggs released by females incubated with NJ05, NJ07 or NJ05 + NJ07 at different concentrations or with vehicle (0.1% DMSO) at different times of exposure from 1 to 3 days. *p < 0.05, **p < 0.01 and *** p < 0.001. For clarity purposes, we show only the highest p-value obtained from the two-way ANOVA test for each time point on all different concentrations tested.
Fig 5.
Micrographs showing alterations on the ultrastructure of the anterior region of Schistosoma mansoni adult male worms exposed to NJ05 or NJ07.
Scanning electron microscopy of worms exposed for 2 days to 25 μM NJ05 (panels 5–8), 25 μM NJ07 (panels 9–12) or vehicle (0.1% DMSO) (panels 1–4). Note that the size scale bar is shown within the black thin line below each image; panels 1 and 2: Control worms (Bar = 500 μm; 100 μm) presented the fully paired couple; panels 3 and 4: Anterior region of control male presented normal oral and ventral suckers tegument (Bar = 50 μm; 30 μm); panels 5 and 6: Anterior region of adult male worm treated with 25 μM NJ05 (Bar = 500 μm; 100 μm); panels 7 and 8: Male oral and ventral suckers presented tegument peeling (Bar = 50 μm); panels 9 and 10: Anterior region of male adult worm treated with 25 μM NJ07 (Bar = 500 μm; 100 μm); panels 11 and 12: Male oral and ventral suckers presented tegument without structural alterations (Bar = 50 μm). f: female; os: oral sucker; vs: ventral sucker; gc: gynecophoral canal; la: lesion area; cp: ciliated papillae.
Fig 6.
Scanning electron micrographs of dorsal and posterior regions of Schistosoma mansoni adult male worm exposed to NJ05 or NJ07.
Worms were exposed for 2 days to 25 μM NJ05 (panels 5–8), 25 μM NJ07 (panels 9–12) or vehicle (0.1% DMSO) (panels 1–4). Panels 1 to 4: Medial (panels 1–3) and posterior (panel 4) regions of control male worm presented normal tegument (Bar = 50 μm; 20 μm; 5 μm; 50 μm). Note that the size scale bar is shown within the black thin line below each image. Panels 5 to 7: Enlarged view of dorsal region of adult male worm treated with NJ05 showing tegument lesions and loss of tubercles (Bar = 50 μm; 20 μm; 5 μm; 50 μm); panel 8: Posterior region of male adult worm treated with NJ05 showing lesion areas (Bar = 50 μm); panels 9 to 11: Dorsal region of male adult worm treated with NJ07 showed bubbles throughout (Bar = 30 μm; 20 μm; 5 μm; 50 μm); panel 12: Posterior region of male adult worm treated with NJ07 (Bar = 50 μm). cp: ciliated papillae; b: blebs; tb: tubercles; la: lesion area.
Fig 7.
Damaged dorsal surface of S. mansoni adult female worms exposed to NJ05 or NJ07.
Female worms were exposed for 2 days to 25 μM NJ05 (panels 5–8), 25 μM NJ07 (panels 9–12) or to vehicle (0.1% DMSO) (panels 1–4) and were observed by scanning electron microscopy; note that the size scale bar is shown within the black thin line below each image. panel 1: Low magnification image of female control worm (Bar = 500 μm). panels 2 to 4: Images of female control worms dorsal region, with normal structural appearance (Bar = 40 μm; 10 μm and 4 μm); panel 5: Low magnification image of female worm exposed to NJ05 (Bar = 500 μm); panels 6 to 8: Middle dorsal region of female presented apparent surface damage with peeling of the tegument membrane (Bar = 50 μm; 10 μm and 5 μm); panel 9: Low magnification image of female worm exposed to NJ07 (Bar = 500 μm); panels 10 to 12: Dorsal region of female worm presented bubbles along the tegument (Bar = 30 μm; 10 μm and 4 μm). p: peeling; la: lesion area; b: blebs.
Fig 8.
Vitellaria and female reproduction gene expression inhibition in adult females upon NJ05 treatment.
Adult couples were treated with NJ05 (25 μM) or with vehicle DMSO (0.1%) for 2 days. The parasites were stored in RNAlater (Ambion) for further processing. All parasite couples were separated and only the females had their RNA extracted followed by cDNA synthesis. The genes measured by RT-qPCR were Smp_131110 (p14), Smp_000430 (Egg Shell Protein (ESP)), Smp_013540 (Tyrosinase 2 (Tyr 2)), Smp_014610 (p48), Smp_000290 (fs800), Smp_055740 (Nanos 2). The geometric mean from two reference genes (Smp_090920 and Smp_123610) was used for normalization with the DCT method. The plotted data is retrieved from DDCT analyses in which the DMSO-treated sample is the control. Significant fold-change in gene expression between DMSO and NJ05 treatment is shown by the numbers inside the brackets. Mean ± SEM of four replicates is shown. Student unpaired parametric two-sided t-test was used, and statistically significant differences are represented by the asterisks. *p ≤ 0.05; **p ≤ 0.01.