TEM of <i>E</i>. <i>multilocularis</i> metacestode exposed to 0.8 μM MMV665807 for 5 days.

<p>(A) and (B) are lower magnification views. LL = laminated layer, Te = tegument, uc = stem cell with large nucleus and nucleolus. Note largely translucent mitochondria in drug treated parasites (mi). Bars in (A) and (B) = 3 μm. (C) = higher magnification view of mitochondria lacking any clearly discernible internal structures. Also note the presence of clearly discernible accumulation of small vesicles within the LL in the close vicinity of the microtriches (arrows). The yellow arrows in (B) and (C) point towards the same location. Bar = 0.3 μm.</p

<i>In vivo</i> MMV665807 treatment in the secondary alveolar echinococcosis mouse model.

<p>(A) Balb/c mice were i.p. infected with metacestodes obtained from <i>in vitro</i> cultures. After 6 weeks of infection, mice were randomly allocated into 4 groups of 6 mice and treated p.o. during 4 weeks. The different treatment groups were: control (no treatment), oil (p.o. gavage of corn oil, 5 days per week), albendazole (ABZ, p.o. gavage of 200 mg/kg ABZ in corn oil, 5 days per week), MMV665807 (p.o. gavage of 100 mg/kg MMV665807 in corn oil, 5 days per week). At the endpoint, mice were euthanized and parasite weight determined. The only effective treatment reducing parasite weight significantly was ABZ. (B) in a second experiment, mice were infected accordingly, and i.p. injection treatment started after 2 weeks of infection. The treatment groups of 8 randomly allocated mice were as follows: control (i.p. injection of DMSO three times per week and p.o. gavage of corn oil 5 days per week), ABZ (i.p. injection of DMSO three times per week and p.o. gavage of 200 mg/kg ABZ in corn oil 5 days per week), MMV665807 (i.p. injection of 100 mg/kg MMV665807 three times per week and p.o. gavage of corn oil 5 days per week) and MMV665807/ABZ (i.p. injection of 100 mg/kg MMV665807 three times per week and p.o. gavage of ABZ in corn oil 5 days per week). After euthanasia, parasite weight was determined. Effective were treatments of ABZ and MMV665807/ABZ. The drug MMV665807 did not lead to any reduction in parasite growth. P values shown in (A) and (B) were calculated by the non-parametric Wilcoxon signed-rank test with Bonferroni-adjustment.</p

Development of a movement-based <i>in vitro</i> screening assay for the identification of new anti-cestodal compounds

<div><p>Intestinal cestodes are infecting millions of people and livestock worldwide, but treatment is mainly based on one drug: praziquantel. The identification of new anti-cestodal compounds is hampered by the lack of suitable screening assays. It is difficult, or even impossible, to evaluate drugs against adult cestodes <i>in vitro</i> due to the fact that these parasites cannot be cultured in microwell plates, and adult and larval stages in most cases represent different organisms in terms of size, morphology, and metabolic requirements. We here present an <i>in vitro</i>-drug screening assay based on <i>Echinococcus multilocularis</i> protoscoleces, which represent precursors of the scolex (hence the anterior part) of the adult tapeworm. This movement-based assay can serve as a model for an adult cestode screen. Protoscoleces are produced in large numbers in Mongolian gerbils and mice, their movement is measured and quantified by image analysis, and active compounds are directly assessed in terms of morphological effects. The use of the 384-well format minimizes the amount of parasites and compounds needed and allows rapid screening of a large number of chemicals. Standard drugs showed the expected dose-dependent effect on movement and morphology of the protoscoleces. Interestingly, praziquantel inhibited movement only partially within 12 h of treatment (at concentrations as high as 100 ppm) and did thus not act parasiticidal, which was also confirmed by trypan blue staining. Enantiomers of praziquantel showed a clear difference in their minimal inhibitory concentration in the motility assay and (R)-(-)-praziquantel was 185 times more active than (S)-(-)-praziquantel. One compound named MMV665807, which was obtained from the open access MMV (Medicines for Malaria Venture) Malaria box, strongly impaired motility and viability of protoscoleces. Corresponding morphological alterations were visualized by scanning electron microscopy, and demonstrated that this compound exhibits a mode of action clearly distinct from praziquantel. Thus, MMV665807 represents an interesting lead for further evaluation.</p></div

TEM of <i>E</i>. <i>multilocularis</i> metacestode exposed to 1.6 μM MMV665807 for 5 days.

<p>Largely non-viable metacestode tissue is seen. (A) shows the separation of the laminated layer (LL) from the tegument (Te) (marked with +), and undifferentiated cells (uc) with a nucleus that is largely consisting of condensed chromatin. Arrows in (A) point towards microtriches, ld in (B) = lipid droplet. (C) shows a metacestode tissue exhibiting large scale necrosis, with the cytoplasm of the tegument being filled with large and small vacuoles of differing content. Arrows in (C) point towards microtriches. Bars in (A) = 4 μm, (B) = 1.5 μm, (C) = 3 μm.</p

Activity assessment of praziquantel and enantiomers on <i>E</i>. <i>multilocularis</i> protoscoleces.

<p><i>E</i>. <i>multilocularis</i> protoscoleces were exposed to various concentrations (100 ppm till 0.0006 ppm, 1 to 3 dilution series) of the PZQ-enantiomers (R)-(-)-PZQ and (S)-(-)-PZQ or the racemic mixture of both (PZQ). After 12 h of incubation, motility was assessed (A) and corresponding pictures of the morphological changes at 0.02 ppm are shown (B). (A) 1% DMSO was set to 100% relative movement and all other drug-responses were calculated accordingly. Note that all forms of PZQ never led to a complete inhibition of protoscolex movement. (R)-(-)-PZQ and the racemic mixture exhibited higher activities than the (S)-(-)-PZQ, with minimal inhibitory concentrations (MICs) indicated by asterisks (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.g001" target="_blank">Fig 1</a> for compound structures and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005618#pntd.0005618.s004" target="_blank">S1 Fig</a> for other measurement time points). Experiments shown here were repeated three times.</p

AlamarBlue vesicle viability assay.

<p>The vesicle viability assay was developed to assess the potential of drugs to kill cells residing within <i>E</i>. <i>multilocularis</i> metacestode vesicles, thus being potentially parasiticidal. Vesicle viability was assessed by alamarBlue assay in triplicates. The drug solvent DMSO served as an internal negative control and was set to 100%. Mefloquine was applied since it is known that this drug is not fully parasiticidal [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004535#pntd.0004535.ref017" target="_blank">17</a>] and it reduced the vesicle viability only at concentrations of 50 μM or higher. The compound MMV665807 was killing cells within vesicles at concentrations as low as 1.6 μM. This underlines the potential parasiticidal activity of the drug MMV665807. Note that the figure depicts average values obtained from three independent measurements and respective standard deviations are shown. All values and SDs are provided in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004535#pntd.0004535.s002" target="_blank">S2 Table</a>.</p

TEM of <i>E</i>. <i>multilocularis</i> metacestodes: Non-treated or treated with 0.4 μM MMV665807.

<p>Non-treated metacestodes (A-C) or metacestodes treated with 0.4 μM MMV665807 for 5 days (D) are shown. (A) is a low magnification view of a section through a non-treated metacestode wall, showing the laminated layer (LL), tegument (Te) and the germinal layer (GL). Clearly visible are undifferentiated stem cells (uc) and the microtriches protruding from the tegument well into the LL (arrows). Bar = 4 μm. Undifferentiated stem cells are shown at higher magnification in (B). mi = mitochondria; nu = nucleus with nucleolus. Bar = 1.5 μm. Higher magnification view of mitochondria are shown in (C). mi = mitochondria, yellow arrows point towards the cristae embedded in an electron matrix. Bar = 0.3 μm. (D) shows a representative micrograph of a metacestode exposed to 0.4 μM MMV665807 for 5 days. Arrows point towards microtriches, mi = mitochondria, mu = muscle cell. Bar = 2.2 μm.</p

Screening cascade for <i>in vitro</i> assessment of drug activity against <i>E</i>. <i>multilocularis</i> metacestodes.

<p>A total of 400 compounds of the Malaria Box were tested first for their ability to induce metacestode damage, employing the PGI assay in singlets at 10 μM. Compounds that showed less than 50% activity of the control were discontinued. 24 active drugs were re-assessed at 1 μM in triplicates. 17 compounds were shown to be inactive and 7 compounds were followed up (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004535#pntd.0004535.g002" target="_blank">Fig 2</a>). The EC₅₀ values of these drugs was determined by PGI assay in triplicates (10–0.01 μM) and drugs with an EC₅₀ value of more than 5 μM were discontinued. In a second major step, host cell toxicity against Reuber rat hepatoma (RH) cells and human foreskin fibroblasts (HFF), both either at a confluent and proliferative state, was assessed by alamarBlue test, and only drugs with a potential therapeutic window were continued. For the remaining MMV665807 the toxicity for isolated and cultured germinal layer cells was assessed (30–0.01 μM) and the IC₅₀ was determined. Since the IC₅₀ against host cells was higher than against germinal layer cells of <i>E</i>. <i>multilocularis</i>, the drug was tested for its ability to reduce the viability of metacestodes by alamarBlue test (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004535#pntd.0004535.g003" target="_blank">Fig 3</a>). The drug MMV665807 that still showed parasiticidal activity at concentrations below the host cell toxicity, finally entered <i>in vivo</i> testing (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004535#pntd.0004535.g007" target="_blank">Fig 7</a>).</p

Drug concentration series of seven compounds against <i>E</i>. <i>multilocularis</i> metacestodes.

<p>Displayed are compounds that exhibited more than 50% activity of the control DB1127 in an initial screen at 10 μM and also activity at 1 μM in the PGI assay against <i>E</i>. <i>multilocularis</i> metacestodes: MMV665794, MMV665941, MMV006319, MMV665807, MMV001318, MMV006169 and MMV011895. Their structures are given below the graph. The seven compounds were tested by PGI assay on <i>E</i>. <i>multilocularis</i> metacestodes in a concentration series ranging from 0.01 to 10 μM in triplicates. DB1127 (10 μM) served as a positive control and was set as 100%. DMSO was the internal negative control that was subtracted from all other values. Note the high activity of MMV665807 down to low concentrations.</p

Assay conditions for motility-based assessment of protoscolex movement.

<p><i>E</i>. <i>multilocularis</i> protoscoleces were purified from metacestodes and activated for 3 h in 10% DMSO. (A) Protoscoleces were distributed into 384-well plates and incubated in various concentrations of DMSO (0, 0.1, 0.3, 1, 3, and 10%) and in 6 replicas each for 12 h, in order to determine the optimal percentage of DMSO to be used for drugs in subsequent tests. The movement per protoscolex was plotted against the respective DMSO concentrations and it shows that protoscolex movement is not significantly reduced up to 3% of DMSO. (B) In order to assess the correlation of protoscolex number per test well and movement, these two parameters were plotted against the total number of protoscoleces per well. A clear linear correlation between number and movement was observed (r = 0.86, R² = 0.75). (C) For determining the optimal assay temperature, protoscoleces were incubated in 1% DMSO at various temperatures (25, 30, 37, and 41°C) with 10 replicas per temperature. As shown, highest movement levels were reached at 37°C, and this was statistically significant. (A-C) The movement of protoscoleces was measured by assessing the changed pixels between two pictures within a 10 seconds interval. (A) and (C) experiments were repeated three times and one exemplary plot is shown.</p