Effect of selected schistosomicidal compounds on <i>S</i>. <i>mansoni</i> worm pairs: gut alterations.

<p>Representative confocal laser microscopy images of the gut of <i>S</i>. <i>mansoni</i> male worms. Worm pairs were treated for 72 hours with the indicated compounds: SmI-1, SmI-11, SmI-291 at 2.5 μM concentration; SmI-233, SmI-251, SmI-290 and SmI-308 at 5 μM concentration and stained with carmine-red. In the gut images, the gastrodermis (ga), carmine-red aggregates (asterisk), and gastrodermis detachment (arrows) are indicated. Scale bars: 50 μm.</p

Discovery by organism based high-throughput screening of new multi-stage compounds affecting <i>Schistosoma mansoni</i> viability, egg formation and production

<div><p>Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus <i>Schistosoma</i>. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of <i>Schistosoma mansoni</i> for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production <i>in vitro</i>.</p></div

Activity of selected hit compounds on adult <i>S</i>. <i>mansoni</i> worms.

<p>Adult male worms (7–8 weeks old) were incubated with the indicated compounds at the concentration of 10 μM (solid circle) and 20 μM (solid square) and viability of parasites scored at different time points (x-axis) as described under methods. DMSO (vehicle, solid triangle) and gambogic acid (solid inverted triangle) were used respectively as negative and positive control. Each point represents the average ± SEM of three independent experiments.</p

Schistosomula HTS screening.

<p>(A) Distribution of Z’ values across all tested plates (128) and Gaussian fitting of binned Z’ values. The dotted vertical line is the 0.5 threshold which is commonly assumed as the lowest value indicating a robust assay (B) Distribution of the tested compound (38, 811) activity expressed as ATP reduction % (death of schistosomula) with respect to DMSO (0%) and gambogic acid 10 μM (100%). The solid curve represents the Gaussian fitting of the binned values. The dotted vertical line is the average plus three times the standard deviation of all compounds (60%), which was set as positivity threshold.</p

Activity on <i>S</i>. <i>mansoni</i> juvenile worms and adult pairs of compounds selected by a pharmacophore approach.

<p>Viability curves of juvenile worms (A) and adult pairs (B) incubated with the indicated compounds using the following concentrations: 2.5 μM (solid square), 5 μM (solid triangle), 10 μM (inverted solid triangle), and 20 μM (solid diamond). DMSO (solid circle) was used as control. The mean data ± SEM of three independent experiments are shown.</p

Effects of SmI-1 and SmI-11 compounds on <i>S</i>. <i>mansoni</i> juvenile worms and adult pairs viability and egg production <i>in vitro</i>.

<p>Viability curves of juvenile worms (4 weeks old) (A) and adult worms pairs (7–8 weeks old) (B) incubated with the SmI-1 and SmI-11 compounds. Worm viability was assessed as described in the methods section. Parasites were cultivated in presence of increasing concentrations of both compounds as follow: 2.5 μM (solid square), 5 μM (solid triangle), 10 μM (solid inverted triangle), and 20 μM (solid diamond). DMSO was used as negative control (solid circle). C) Total egg counts laid by parasites treated during 72 hours with different sub-lethal doses of SmI-1 and SmI-11 normalized to worm couples. The mean data ± SEM of three independent experiments are shown. The levels of statistical significance are indicated above bars; *p-value < 0.05, ****p-value < 0.0001, Student's <i>t</i> test.</p

Activity on schistosomula of compounds selected by a pharmacophore approach.

<p>Compounds were grouped based on the parent compound. Compounds within the red frame were identified based on the SmI-10 pharmacophore, compounds within the orange frame were identified based on the SmI-1 pahrmacophore, and the compound in the black frame was identified by the SmI-11 pharmacophore. For each compound dose response curves on schistosomula viability assay are reported. The % ATP reduction (schistosomula death) plotted on the y-axis is normalized between DMSO (0%) and gambogic acid 10 μM (100%). Each point represents the average and standard deviations of three independent experiments.</p

Activity on the production of eggs of compounds selected by a pharmacophore approach.

<p>A) Histograms showing the total egg counts normalized to worm couples treated for 72 hours with sub-lethal doses of the indicated compounds. The levels of statistical significance are indicated above bars: ** p-values ≤ 0.01, *** p-values ≤0.001, Student's <i>t</i> test. B) Representative images of eggs, oocytes (oc), spermatozoa (sp), vitelline cells (vc), eggshell fragments (ef), and abnormal eggs (ae) in the tissue culture medium of worm couples treated with DMSO or 5 μM of SmI-290, or SmI-308 at 24 and 72 hours. Tissue culture medium and compounds were freshly replaced every 24 hours. Scale bars: 100 μm.</p

Activity on adult <i>S</i>. <i>mansoni</i> worms of compounds selected by a pharmacophore approach.

<p>Viability curves of adult male worms (7–8 weeks old) incubated with the indicated compounds at the concentration of 10 μM (solid square) and 20 μM (solid triangle). The viability of parasites scored at different time points (x-axis) as described under methods. DMSO (solid circle) was used as control. Each point represents the average ± SEM of three independent experiments.</p

Effect of selected schistosomicidal compounds on the phenotype of <i>S</i>. <i>mansoni</i> worm pairs: Female reproductive organs alterations.

<p>Representative confocal laser microscopy images of <i>S</i>. <i>mansoni</i> female reproductive system (ootype, ovary, oviduct and vitellarium). Worm pairs were treated for 72 hours with the indicated compounds: A) SmI-1 and SmI-11 at 2.5 μM concentration; SmI-233 and SmI-251 at 5 μM concentration; B) SmI-290 and SmI-308 at 5 μM concentration and SmI-291 at 2.5 μM concentration (ootype, oviduct and vitellarium images) and 5 μM concentration (ovary). Worm pairs were stained with carmine-red as described in the methods section. In Figure 10A and 10B immature oocytes (io), mature oocytes (mo), uterus (ut), ootype (ot), vitelline duct (vd), oviduct (od), vitello-oviduct (vd/od). Arrows indicate degenerated cells and asterisk mature oocytes engulfment within the oviduct. Scale bars: 50 μm.</p