A new semi-automated, motility-based screening assay for discovery of compounds with activity against the juvenile stage of Fasciola hepatica

Fasciola hepatica is a trematode parasite responsible for fasciolosis, a liver disease that affects humans and livestock worldwide. Together with other food-borne trematode infections, fasciolosis is considered a neglected tropical disease. Further, it imposes substantial agricultural losses due to infections in ruminants. No vaccine is currently available, and control heavily relies on drug treatment, especially with triclabendazole (TCBZ). However, the intensive use of TCBZ over the past four decades has led to increasing rates of treatment failures and the emergence of drug-resistant parasites. Therefore, the identification of new treatment options is an urgent priority. The currently available toolset for drug screening, however, is limited. To address this need, we established a novel, semi-automated, standardized, and objective screening assay based on motility monitoring of newly excysted juveniles using microscopic live imaging. The assay was validated by testing a panel of ten compounds with known anthelmintic properties, amongst them TCBZ (IC50: 1.5 µM) and the new activator of the F. hepatica transient receptor potential melastatin (TRPMPZQ) ion channel, benzamidoquinazolinone (IC50: 1.05 µM). In addition to these two compounds with known activity against F. hepatica, three compounds were identified as particularly promising with a fast onset of action and IC50 values in the nanomolar range: the salicylanilides MMV665807 (IC50: 44 nM), niclosamide (IC50: 32 nM), and its ethanolamine salt, niclosamide ethanolamine (IC50: 9 nM). Complementary live/dead staining revealed that only TCBZ displayed parasiticidal activity, while the other compounds, although leading to parasite paralysis, did not lead to parasite death within 72 hours. Scanning electron microscopy of drug treated parasites did not reveal any significant damage at concentrations corresponding to the IC50s, but strong phenotypes were visible at 20 µM. The presented motility assay provides a robust method for the discovery of novel anthelmintic compounds and facilitates the ongoing effort to combat fasciolosis.