Fig 1.
In vitro drug screening cascade for the identification of active compounds against E. multilocularis and E. granulosus s.s.
The drug screening cascade enables the identification of active compounds against E. multilocularis or E. granulosus s.s. metacestode vesicles, protoscoleces and GL cells via different in vitro assays. First, an overview screening is performed with metacestode vesicles to measure the direct impact on the physical integrity of metacestode vesicles via PGI assay [61], and a potential impairment of viability via vesicle viability assay [32]. Next, the damaging effect against protoscoleces is tested via motility assay [31], followed by cytotoxicity measurements in mammalian cells via alamar blue assays [61]. Finally, the parasiticidal potential of drug candidates against GL cells of the parasite are studied by measuring ATP levels [61]. Compounds that show parasiticidal activity against metacestode vesicles and GL cells, as well as a low toxicity against host cells, may be further studied for structure activity relationship via PGI assay and their mode of action via various specific in vitro assays [26,61]. Finally, promising compounds are tested in experimentally infected mice for their activity in vivo [32,63]. Figure adapted from [62].
Fig 2.
In vitro cultured E. granulosus s.s. metacestode vesicles.
E. granulosus s.s. metacestode vesicles were generated upon in vitro culture of GL from ex vivo CE cysts. A: Metacestode vesicles appeared after three weeks as indicated by arrows. B: After five weeks, vesicles generated a visible laminated layer as indicated by arrows. C: Metacestode vesicles after ten months of culture. D: Metacestode vesicles after 20 months of culture. Scale bars represent one mm in A and B or five mm in C and D. E and F: TEM of E. granulosus s.s. metacestode vesicles after four months of culture. LL = laminated layer; GL = germinal layer; gsc = glycogen storage cell; m = mitochondrion, nuc = nucleus. Note the presence of very short microtriches (arrows), and the absence of a discernible tegument at the LL-GL interface. Scale bars represent 4 μm and 8 μm in E and F, respectively.
Fig 3.
Assessment of drug efficacy on E. multilocularis and E. granulosus s.s. metacestode vesicles by PGI assay and metacestode vesicle viability assay.
Representative images of E. multilocularis and E. granulosus s.s. metacestode vesicles treated with the negative control 0.1% DMSO, the positive control 0.1% TX-100, or different drugs after five days (A) and twelve days (B) are shown. PGI release of E. multilocularis and E. granulosus s.s. metacestode vesicles into culture supernatant was measured after five days (C) and twelve days (D) and is shown in relation to the positive control 0.1% TX-100. Metacestode vesicle viability was measured after twelve days and is shown in relation to the negative control 0.1% DMSO (E). The activities of ABZ (albendazole), BPQ (buparvaquone), MEF (mefloquine), MMV-X (MMV665807), MPT (monepantel), NIC (niclosamide) and NTZ (nitazoxanide) were assessed in vitro against metacestode vesicles of E. multilocularis and E. granulosus s.s. Drug activity was calculated in percentage as relative PGI release compared to the positive control 0.1% TX-100. The drugs were tested at 40 μM concentrations, except MMV-X and NIC, which were tested at 1 μM. The metacestode vesicles were incubated for five and twelve days under a humid microaerobic atmosphere (85% N2, 10% CO2, 5% O2) and tests were performed in triplicates per condition. Shown are mean values and standard deviations of three independent experiments. Relative PGI release of E. multilocularis and E. granulosus s.s. metacestode vesicles was compared and significance is shown according to bonferroni-corrected p values of p <0.05 obtained using multiple two-tailed students t-tests assuming equal variance.
Fig 4.
Effects of drugs on E. multilocularis and E. granulosus s.s. protoscolex motility.
The activities of PZQ (praziquantel) racemate (B), as well as of (R)-(-)-PZQ (C), (S)-(-)-PZQ (D) and various standard drugs (ABZ (albendazole) (E), MMV-X (MMV665807) (F), MPT (monepantel) (G), NIC (niclosamide) (H), and NTZ (nitazoxanide) (I)) were assessed. PZQ and its enantiomers were tested at concentrations of 100 to 0.0006 ppm (320 to 0.0018 μM). The other drugs were tested at concentrations from 100 to 0.4 ppm which corresponded to 377 to 1.6 μM for ABZ, 317 to 1.3 μM for MMV-X, 211 to 0.9 μM for MPT, 306 to 1.3 μM for NIC and 325 to 1.3 μM for NTZ. The relative motility is shown in comparison to the solvent control (1% DMSO). The morphology of protoscoleces treated with PZQ and its enantiomers is shown for 0.015 ppm (0.05 μM) and for protoscoleces treated with the other drugs, the morphology is shown for 33 ppm which corresponded to 126 μM for ABZ, 106 μM for MMV-X, 70 μM for MPT, 102 μM for NIC and 108 μM for NTZ, respectively. All concentrations were tested in six replica. Shown are mean values and standard deviations of three independent experiments. Significant bonferroni-corrected p values with p<0.05 are shown that were obtained using students t-test comparing relative movement between E. multilocularis and E. granulosus s.s. protoscoleces for each individual drug concentration. EC50 values of the three independent experiments are shown in S1 Table as mean values and SD in ppm and μM.
Fig 5.
Metacestode vesicle formation from E. granulosus s.s. GL cells.
E. granulosus s.s. GL cells were cultured in cDMEM supplemented with 100 μM serotonin with three medium changes a week under microaerobic conditions. Micrographs were taken after five days (A), twelve days (B) and 26 days (C). Scale bars represent 100 μm. Red arrows highlight the position of the metacestode vesicle.
Fig 6.
Efficacy of drugs against E. multilocularis and E. granulosus s.s. GL cells.
The activities of ABZ (albendazole), BPQ (buparvaquone), MEF (mefloquine), MMV-X (MMV665807), MPT (monepantel), NIC (niclosamide) and NTZ (nitazoxanide) were assessed for E. multilocularis and E. granulosus s.s. primary cells. Representative images are shown with scale bars representing 200 μm (A), relative cell viability after five days of incubation with the compounds is shown in comparison to the solvent control (0.1% DMSO) (B). Drugs were tested at 40 μM concentrations, except MMV-X and NIC, which were tested at 1 μM. For each experiment, 15 AU of primary cells of either E. multilocularis or E. granulosus s.s. were incubated in cDMEM containing the respective compounds. The plate was incubated for 5 days under a humid microaerobic atmosphere (85% N2, 10% CO2, 5% O2) and performed in four replica per condition. Shown are mean values of three independent experiments. Significant bonferroni-corrected p values with p<0.05 are shown that were obtained using multiple students t-test comparing relative cell viability between E. multilocularis and E. granulosus s.s. GL cells for the different drugs.