Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load

Activities of fenbendazole in comparison with albendazole against Echinococcus multilocularis metacestodes in vitro and in a murine infection model.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE

Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors

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

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

<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

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