<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

In vivo treatment of secondary <i>E. multilocularis</i> infected Balb/c mice with BTZ reduces parasite weight.

<p>Balb/c mice were i.p. infected with in vitro-cultured <i>E. multilocularis</i> parasite material. After 6 weeks, treatment was performed for 6 weeks (5 days a week) with 2×5 mice in each treatment group. Controls received honey/CMC p.o. and PBS i.p. ABZ received 200 mg/kg ABZ in honey/CMC and PBS i.p. BTZ received honey/CMC p.o. and 0.7 mg/kg BTZ i.p. once a week for three weeks, then 0.5 mg/kg twice a week for another three weeks. ABZ/BTZ received ABZ p.o. and BTZ i.p. as stated above. After euthanization parasite material was resected (A) and weighed (B). A, macroscopical assessment showed proliferating metacestode masses with many big and small vesicles (see black arrows) in the control group. All treated groups showed less metacestodes, especially less proliferating ones and more white and encapsulated, small cysts (see white arrows). B, parasite weight visualized by boxplot. Statistical analysis of log-transformed data confirmed a highly significant parasite mass reduction with ABZ and ABZ/BTZ treatment.</p

Proteasome activity in <i>E. multilocularis</i> cell extracts can be inhibited by BTZ.

<p>Chymotrypsin-like activity of the <i>E. multilocularis</i> proteasome was shown in cell extracts from in vitro-cultured metacestodes by applying the fluorogenic substrate SLLVT-AMC in solution (A) and in gel (B). Addition of BTZ led to dose-dependent inhibition of this activity. A, in solution proteasome assay with and without Halt protease inhibitor (to inhibit other proteases). As a control DMSO was added to same amounts as with the drug and the activity calculated as percentage from the DMSO control. This experiment was performed in technical quadruplicates and was repeated three times, which led to similar results each time. B, in gel assay to detect chymotrypsin-like activity in <i>E. multilocularis</i> cell extracts. Bovine chymotrypsin was used as positive control (C, 5 ng per lane), <i>E. multilocularis</i> extract was loaded at 25 µg. On the left the gel was incubated with DMSO, on the right with BTZ at 10 µM. C, Western blot was performed to detect the <i>E. multilocularis</i> proteasome subunit beta 5 (EmPSMB5). Note that this protein migrates at the same apparent molecular mass as the active band in B that could be inhibited by BTZ. Experiments B and C were repeated both twice leading to the same results.</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

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

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

Activity of the most active drugs of the FDA library against <i>E. multilocularis</i> metacestodes in vitro.

<p>The seven most active drugs (more than 50% damage of the control DB1127 at 20 µM after 5 days; BTZ, sorafenib tosylate, crystal violet, candesartan cilexetil, nitazoxanide, amlodipine besylate and axitinib) of the FDA drug library screen were further tested in triplicates by PGI assay at different concentrations (20, 10, 5, 1, 0.1 µM) and for their anti-metacestode activity in vitro. As a positive control, DB1127 was applied at 20 µM and the different drug activities are expressed as percentage of the positive control. DMSO served as a negative control at same dilutions as the drugs and was subtracted from all values. Note the high activity of BTZ down to the concentration of 0.1 µM.</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

Accumulation of ubiquitinated proteins upon <i>E. multilocularis</i> metacestode treatment with BTZ.

<p><i>E. multilocularis</i> metacestodes were treated in vitro with BTZ (10 µM) for 24 h in triplicates and proteins extracted and visualized by Western blot (A, B). Treatment with equal amounts of DMSO served as a negative control. GAPDH was used as sample loading control (A), the anti-ubiquitin antibody detected ubiquitinated proteins in each sample; M = Marker (B). Semiquantitative analysis of ubiquitinated proteins was performed in Image J and relative protein abundance shown in relation to the untreated controls (C). This experiment was performed twice and lead to similar results of 3.5 times accumulation of ubiquitinated proteins upon BTZ treatment.</p

Amino acid sequence alignment of the proteasome subunit 5 beta of <i>Saccharomyces cerevisiae, Homo sapiens</i> and <i>E. multilocularis</i>.

<p>The amino acids of the active core of PSMB5 are highlighted in light grey, amino acids involved in BTZ binding are highlighted in dark grey (based on <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003352#pntd.0003352-Gille1" target="_blank">[22]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003352#pntd.0003352-Groll1" target="_blank">[23]</a>). One amino acid is both part of the active core and involved in BTZ binding (T67 of <i>E. multilocularis</i> sequence). One amino acid involved in BTZ binding shows a conservative replacement (T→S88). The <i>E. multilocularis</i> PSMB5 was 52% identical to <i>S. cerevisiae</i> PSMB5 and 61% identical to the human ortholog. The alignment was performed in ClustalW2.1.</p