In vitro screening of the open-access Pandemic Response Box reveals ESI-09 as a compound with activity against Echinococcus multilocularis

Abstract

Alveolar echinococcosis (AE) is a life-threatening disease caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis, primarily in the liver. Current drug treatments rely on benzimidazoles, which are not parasiticidal, requiring life-long therapy with significant side effects. Therefore, novel drug treatments are urgently needed. Drug repurposing offers a strategy to identify novel therapies against the neglected disease AE. We report on the in vitro screening of the Pandemic Response Box, an open-access compound library composed of 400 drug-like molecules assembled by Medicines for Malaria Venture (MMV) and the Drugs for Neglected Disease Initiative (DNDi), against E. multilocularis. An overview screen at 10 μM using the metacestode vesicle damage-marker release assay (based on release of phosphoglucose isomerase, PGI) and metacestode vesicle viability assay (based on ATP measurement) identified 37 active compounds. Reassessment in triplicates resulted in five active compounds (alexidine, carbendazim, ESI-09, MMV1581545, oxfendazole) displaying anti-metacestode activity. The parasiticidal activity of these five compounds was evaluated by ATP measurement in germinal layer cells. One compound, ESI-09, acted specifically against E. multilocularis (IC₅₀ on metacestode vesicles 6.06 ± 3.18 μM by PGI release assay and 2.09 ± 0.56 μM by metacestode vesicle viability assay as well as an IC₅₀ of 2.45 ± 0.86 μM on germinal layer cells) with a broad therapeutic window when compared to mammalian cell toxicity. Further experiments applying Seahorse technology and tetramethylrhodamine ethyl ester (TMRE) assay revealed that ESI-09 acts as a mitochondrial uncoupler in parasite cells. However, transmission electron microscopy showed no significant ultrastructural changes in parasite mitochondria, though increased secretion of extracellular vesicle-like structures between the tegument and the laminated layer was observed. In summary, screening of the Pandemic Response Box identified ESI-09 as a potential drug candidate for the treatment of AE. Further experiments are needed to evaluate the efficacy of ESI-09 in vivo.

Keywords: Alveolar echinococcosis; DNDi; Drug repurposing; Echinococcus mutlilocularis; MMV; Mitochondrial uncoupler.

Graphical abstract

Fig. 1

Overview and confirmation screen of the Pandemic Response Box againstE. multilocularismetacestode vesicles. Compounds were tested at 10 μM and metacestode vesicles were incubated for 12 days at 37 °C under a humid, 5 % CO₂ atmosphere. (A) Overview screen was done in singlets. Red dashed lines indicate cut-off for active compounds (PGI release ≥20 % and viability ≤30 %). Purple points represent active compounds. (B) Confirmation screen of the 37 initial hits. Red dashed lines indicate cut-off for active compounds (PGI release ≥20 % and Viability ≤30 %). Shown are means ± SD. (C) 2D structures of active compounds. Structures were generated using ChemDraw.

Fig. 2

Dose-response curves of the active compounds on GL cells. Compounds (alexidine, ESI-09 and MMV1581545) were tested on isolated E. multilocularis GL cells at varying concentrations. Cells were incubated for 5 days under a microaerobic atmosphere at 37 °C. (A) Representative pictures of GL cell aggregates incubated with the different compounds and concentrations as indicated. (B) Viability of GL cells was tested using CellTiter-Glo® in quadruplicates and plotted relative to the solvent control DMSO. Shown are means ± SD and the non-linear regression models. Plot shows one representative experiment. The experiment was performed three times independently (S5 Fig).

Fig. 3

Dose-response curves for ESI-09 tested on whole metacestode vesicles. The effect of ESI-09 was tested at varying concentrations. Metacestode vesicles were incubated for 12 days under a humid, 5 % CO₂ atmosphere at 37 °C and measurements were done in triplicates. Shown are means ± SD and the fitted non-linear regression model. Plot shows one representative experiment. This experiment was performed three times independently (S6 Fig.). (A) Representative pictures of metacestode vesicles treated with ESI-09 at different concentrations as indicated. Note the clear detachment of the GL from the laminated layer, which is visible from 13.3 μM on. (B) Damage-marker release relative to the total damage control Tx-100 measured after 12 days by PGI release assay. (C) Viability of metacestode vesicles relative to the solvent control DMSO measured after 12 days by metacestode vesicle viability assay.

Fig. 4

ESI-09 acts as a mitochondrial uncoupler inE. multilocularisGL cells. Oxygen consumption rate (OCR) was measured using Seahorse XFp analyzer in technical duplicates. (A) Relative OCR increase upon injection of either DMSO, 2 μM ESI-09 or 0.5 μM FCCP. Shown are individual data points of three independent experiments in the box plot. Pairwise comparisons were performed using the Wilcoxon Rank Sum Test with Bonferroni correction for multiple comparisons. Significance levels: 0.001 < p ≤ 0.01 (∗∗), p > 0.05 (ns). (B) TMRE fluorescence intensity was measured via confocal microscopy. GL cells were either treated with 10 μM ESI-09, FCCP or the solvent control DMSO. Representative microscopy pictures of maximal intensity z-stacks. (C) TMRE fluorescence intensity was measured via confocal microscopy. GL cells were either treated with 10 μM ESI-09, FCCP or the solvent control DMSO. Quantification of TMRE fluorescence signal normalized to GL cell aggregate area. Shown are single values of 30 individual aggregates. The purple crossbar represents the mean value, while the vertical bar represents the SD. Pairwise comparisons were performed using the Wilcoxon Rank Sum Test with Bonferroni correction for multiple comparisons. Significance levels: p ≤ 0.0001 (∗∗∗∗).

Fig. 5

Effects of ESI-09 on metacestode vesicle ultrastructure.E. multilocularis metacestode vesicles were treated either with 1 μM of ESI-09 or the solvent control DMSO for 24 h. Ultrastructural morphology of metacestode vesicles treated with (A) DMSO or (B) ESI-09. GL: germinal layer, LL: laminated layer, TE: tegument. gsc: glycogen storage cells, mc: muscle cell, mito: mitochondria, mt: microtriches. (C) Yellow arrows indicate electron dense lipid droplets observed after ESI-09 treatment. (D) Phagosome-like structures indicated by yellow arrows upon ESI-09 treatment. (E) ESI-09-induced accumulation of extracellular vesicle-like structures at the tegument-laminated layer interface indicated by yellow arrows. (F) Yellow arrows show electron-dense mitochondria with visible cristae upon ESI-09 treatment.

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