Development of a movement-based in vitro screening assay for the identification of new anti-cestodal compounds

摘要

Intestinal cestodes are infecting millions of people and livestock worldwide, but treatment is mainly based on one drug: praziquantel. The identification of new anti-cestodal compounds is hampered by the lack of suitable screening assays. It is difficult, or even impossible, to evaluate drugs against adult cestodes in vitro due to the fact that these parasites cannot be cultured in microwell plates, and adult and larval stages in most cases represent different organisms in terms of size, morphology, and metabolic requirements. We here present an in vitro-drug screening assay based on Echinococcus multilocularis protoscoleces, which represent precursors of the scolex (hence the anterior part) of the adult tapeworm. This movement-based assay can serve as a model for an adult cestode screen. Protoscoleces are produced in large numbers in Mongolian gerbils and mice, their movement is measured and quantified by image analysis, and active compounds are directly assessed in terms of morphological effects. The use of the 384-well format minimizes the amount of parasites and compounds needed and allows rapid screening of a large number of chemicals. Standard drugs showed the expected dose-dependent effect on movement and morphology of the protoscoleces. Interestingly, praziquantel inhibited movement only partially within 12 h of treatment (at concentrations as high as 100 ppm) and did thus not act parasiticidal, which was also confirmed by trypan blue staining. Enantiomers of praziquantel showed a clear difference in their minimal inhibitory concentration in the motility assay and (R)-(-)-praziquantel was 185 times more active than (S)-(-)-praziquantel. One compound named MMV665807, which was obtained from the open access MMV (Medicines for Malaria Venture) Malaria box, strongly impaired motility and viability of protoscoleces. Corresponding morphological alterations were visualized by scanning electron microscopy, and demonstrated that this compound exhibits a mode of action clearly distinct from praziquantel. Thus, MMV665807 represents an interesting lead for further evaluation.