Fig 1. Screening cascade for in vitro assessment of drug activity against E. multilocularis metacestodes.

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 Fig 2). 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 E. multilocularis, the drug was tested for its ability to reduce the viability of metacestodes by alamarBlue test (see Fig 3). The drug MMV665807 that still showed parasiticidal activity at concentrations below the host cell toxicity, finally entered in vivo testing (see Fig 7).