Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays.

BACKGROUND: Schistosomiasis is responsible for a tremendous public health burden, yet only a single drug, praziquantel, is available. New antischistosomal treatments should therefore be developed. The accuracy, speed and objectivity of in vitro drug screening depend on the assay read-out. Microscopy is still the current gold standard and is in need of updating to an automated format. The aim of the present study was to investigate a panel of fluorescence/luminescence dyes for their applicability as viability markers in drug sensitivity assays for Schistosoma mansoni schistosomula. METHODS: A search for available viability and cytotoxicity marker assays and dyes was carried out and a short-list of the most interesting candidates was created. The selected kits and dyes were tested on S. mansoni Newly Transformed Schistosomula (NTS), first to assess whether they correlate with parasite viability, with comparatively low background noise, and to optimise assay conditions. Markers fulfilling these criteria were then tested in a dose-response drug assay using standard and experimental drugs and those for which an IC50 value could be accurately and reproducibly calculated were also tested on a subset of a compound library to determine their hit-identification accuracy. RESULTS: Of the 11 markers selected for testing, resazurin, Vybrant® and CellTiter-Glo® correlated best with NTS viability, produced signals ≥ 3-fold stronger than background noise and revealed a significant signal-to-NTS concentration relationship. Of these, CellTiter-Glo® could be used to accurately determine IC50 values for antischistosomals. Use of CellTiter-Glo® in a compound subset screen identified 100% of hits that were identified using standard microscopic evaluation. CONCLUSION: This study presents a comprehensive overview of the utility of colorimetric markers in drug screening. Our study demonstrates that it is difficult to develop a simple, cheap "just add" colorimetric marker-based drug assay for the larval stage of S. mansoni. CellTiter-Glo® can likely be used for endpoint go/no go screens and potentially for drug dose-response studies.

Activity of praziquantel enantiomers and main metabolites against Schistosoma mansoni.

A racemic mixture of R and S enantiomers of praziquantel (PZQ) is currently the treatment of choice for schistosomiasis. Though the S enantiomer and the metabolites are presumed to contribute only a little to the activity of the drug, in-depth side-by-side studies are lacking. The aim of this study was to investigate the in vitro activities of PZQ and its main metabolites, namely, R- and S-cis- and R- and S-trans-4'-hydroxypraziquantel, against adult worms and newly transformed schistosomula (NTS). Additionally, we explored the in vivo activity and hepatic shift (i.e., the migration of the worms to the liver) produced by each PZQ enantiomer in mice. Fifty percent inhibitory concentrations of R-PZQ, S-PZQ, and R-trans- and R-cis-4'-hydroxypraziquantel of 0.02, 5.85, 4.08, and 2.42 µg/ml, respectively, for adult S. mansoni were determined in vitro. S-trans- and S-cis-4'-hydroxypraziquantel were not active at 100 µg/ml. These results are consistent with microcalorimetry data and studies with NTS. In vivo, single 400-mg/kg oral doses of R-PZQ and S-PZQ achieved worm burden reductions of 100 and 19%, respectively. Moreover, worms treated in vivo with S-PZQ displayed an only transient hepatic shift and returned to the mesenteric veins within 24 h. Our data confirm that R-PZQ is the main effector molecule, while S-PZQ and the metabolites do not play a significant role in the antischistosomal properties of PZQ.

Orally active antischistosomal early leads identified from the open access malaria box.

BACKGROUND: Worldwide hundreds of millions of schistosomiasis patients rely on treatment with a single drug, praziquantel. Therapeutic limitations and the threat of praziquantel resistance underline the need to discover and develop next generation drugs. METHODOLOGY: We studied the antischistosomal properties of the Medicines for Malaria Venture (MMV) malaria box containing 200 diverse drug-like and 200 probe-like compounds with confirmed in vitro activity against Plasmodium falciparum. Compounds were tested against schistosomula and adult Schistosoma mansoni in vitro. Based on in vitro performance, available pharmacokinetic profiles and toxicity data, selected compounds were investigated in vivo. PRINCIPAL FINDINGS: Promising antischistosomal activity (IC50: 1.4-9.5 µM) was observed for 34 compounds against schistosomula. Three compounds presented IC50 values between 0.8 and 1.3 µM against adult S. mansoni. Two promising early leads were identified, namely a N,N'-diarylurea and a 2,3-dianilinoquinoxaline. Treatment of S. mansoni infected mice with a single oral 400 mg/kg dose of these drugs resulted in significant worm burden reductions of 52.5% and 40.8%, respectively. CONCLUSIONS/SIGNIFICANCE: The two candidates identified by investigating the MMV malaria box are characterized by good pharmacokinetic profiles, low cytotoxic potential and easy chemistry and therefore offer an excellent starting point for antischistosomal drug discovery and development.