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

Abstract

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.

Nematocidal Effects of a Coriander Essential Oil and Five Pure Principles on the Infective Larvae of Major Ovine Gastrointestinal Nematodes In Vitro

“…The inhibition of larval motility, as an indicator of worm viability after exposure to molecules or substances, is generally used in drug screening [23] and as a gold standard for testing anthelmintics [54]. However, this assay seems to be impractical especially when evaluating numerous compounds or concentrations [24].…”

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis

“…and other parasites, is not active against the metacestode stage of E. multilocularis , neither in vivo ( Vanparijs, 1990 ), nor in vitro, as confirmed in this study. This could be explained by the fact that praziquantel causes paralysis of the parasite musculature, which then only affects actively moving, adult worms but not sessile metacestode larvae ( Ritler et al, 2017 ; Vale et al, 2017 ). The antifungal agent amphotericin B (MMV689000) was shown to destroy E. multilocularis metacestodes in vitro at 2.7 μM ( Reuter et al, 2003 , 2010 ).…”

Pharmacophore-Based Virtual Screening Toward the Discovery of Novel Anti-echinococcal Compounds

“…Recently, algorithms and imaging systems have been developed to measure the effects of drugs on the activities of E. multilocularis protoscoleces (PSCs). This advance has greatly increased screening efficiencies and reduced the requirement for experienced researchers; thus, it is considered an ideal screening method (Ritler et al, 2017). However, this improved test method has not substantially change the anti-echinococcal drug development pathway.…”