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

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

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.; 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.; 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.; 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

Accelerating antischistosomal drug discovery : preclinical studies of antimalarials, synthetic peroxides and praziquantel derivatives

Schistosomiasis is one of the most devastating parasitic diseases in tropical countries and remains a major public health problem, especially in Sub-Saharan Africa. It is caused by blood flukes of the species Schistosoma. Schistosoma  haematobium, S. mansoni and S. japonicum are the main species responsible for the largest number of cases. Chronic schistosomiasis is caused by the immunological response to eggs trapped in tissue and organs. In the case of S. mansoni, eggs get trapped in the gut wall and liver, leading to severe tissue destruction. Treatment with praziquantel (PZQ) is the mainstay of morbidity control. PZQ is safe and efficacious, but it is the only available drug. Moreover, it has a major efficacy limitation due to lacking efficacy on juvenile Schistosoma stages. Hence new backup drugs are strongly required. Antischistosomal drug discovery has been neglected for a long time. As a consequence, the antischistosomal drug pipeline is empty. Technical innovations for antischistosomal drug screening and new promising drug candidates are urgently needed. My PhD thesis had two major aims. The first was to improve the drug screening process by evaluating new assays and readouts and by refining the screening cascade. The second was to further investigate known lead compounds and to identify entirely new drug candidates and chemical scaffolds. A literature review conducted at the beginning of my PhD thesis revealed that clinical trials conducted in pediatrics, are mainly concerned with antimalarials and only 3 % are dealing with schistosomiasis. Especially the field of PK trials is completely neglected and the importance of investigating the relationship of infections and PK changes was emphasized. The common blood-feeding characteristic of schistosomes and Plasmodia has led to studies with antimalarial drugs against schistosomes in recent years. Amongst different chemical structures, mefloquine (MFQ), a 4-quinolinemethanol, and the artemisinins, with their distinct peroxidic scaffold, qualified as leads for antischistosomal drug discovery. The in vitro and in vivo antischistosomal potential of selected MFQ-related arylmethanols was characterized. Furthermore the role of various iron sources in in vitro drug activity was investigated to get insights into the mode of action (MOA). Pharmacokinetic (PK) and pharmacodynamic (PD) properties of lead candidates were explored in S. mansoni infected and uninfected mice. The class of 4-quinolinemethanols revealed the best in vitro activity (IC50s < 3.5 µM) against adult schistosomes. A ten-fold increase in activity was observed for the two lead 4-quinolinemethanols (MFQ, WR7930) when incubated in the presence of hemoglobin. High worm burden reductions (83 - 100%) were observed for EP (4-pyridinemethanol) and WR7930 (4-quinolinemethanol) in mice harboring adult S. mansoni or S. haematobium. EP and MFQ were selected for further PK investigations. A HPLC-UV method was successfully validated to measure the two drugs of interest simultaneously within mouse plasma. The migration of schistosomes to the liver after treatment with active drugs, known as hepatic shift, was delayed for both drugs (72 - 168 h). Dramatic changes in the drug disposition of MFQ and EP were triggered by the S. mansoni infection. Increased AUCs and half-lives led to slowed drug clearance. Driven by these promising antischistosomal properties of antimalarials the MMV Box, containing 200 drug-like and 200 probe-like compounds, was investigated. Two entirely new chemical scaffolds, the diarylureas and the dianilinoquinoxalines, presented excellent in vitro activity (IC50: 0.8 μM) as well as moderate in vivo worm burden reductions (WBR: 40.8 - 52.5 %) following single oral drug administration in S. mansoni infected mice. To pursue the active peroxidic scaffold further, various promising peroxide classes- ozonides (OZs), 3-alkoxy-1, 2-dioxolanes, tetraoxanes, tricyclic monoperoxides and alphaperoxides- were tested against juvenile and adult S. mansoni stages. Additionally the roles of iron and the peroxidic core in drug activity were evaluated. Promising in vitro activity on both stages was observed for the alkoxydioxolanes. However, only moderate, non-significant activity was observed in vivo. Iron sources did not alter activity on schistosomes, supporting an iron-independent MOA. Non-peroxidic alkoxydioxolane analogues lacked activity against both parasites, underlining the necessity of a peroxide functional group. Investigations on the three new peroxidic classes (tetraoxanes, tricyclic monoperoxides and alphaperoxides) aimed to get more insights on the structural needs of peroxidic drugs for antischistosomal activity. High in vitro activitiy was revealed on the schistosomular stage, but decreased susceptibilities were observed on the adult stage. One tetraoxane and one tricyclic monoperoxide presented good in vivo activity against adult Schistosoma infections in mice but lacked efficacy on the juvenile infection in vivo. A non-iron dependent activation was observed for these classes as well. Both lead candidates represent new chemical scaffolds but come along with cytotoxicity limitations. Furthermore both compounds are at the very early stage of drug development, and extensive further research is needed for successful drug development. The most promising peroxidic lead candidate was elucidated amongst the OZs, OZ418, with excellent in vivo activity in both juvenile and adult Schistosoma infections. Additionally we were interested in the antischistosomal activity of PZQ derivatives resulting from novel organometallic derivatization strategies. However organometallic ferrocenyl PZQ derivatives presented only moderate in vitro effects against adult worms. The derivatization of PZQ with chromium moieties led to promising in vitro results but has so far shown to be inefficacious in vivo. During the process of evaluating this broad range of chemical scaffolds and lead candidates we worked on the improvement of the screening cascade, identifying new parameters resulting in improved in vitro - in vivo correlation, evaluating cut-offs and their impact on hit decision as well as investigating new readout tools for drug screenings assays. During the work of this thesis an in vitro motility assay based on S. haematobium schistosomula was successfully developed. Colorimetric markers were evaluated as an alternative readout technique, but no promising results were achieved so far. In conclusion, MFQ and EP are interesting antischistosomal lead candidates, however their extensive half-lives and slow clearance might be an issue regarding the strict safety profile. In general antimalarials presented an excellent starting point for antischistosomal drug discovery. Amongst the peroxidic classes, OZ418 showed great potential for a good lead candidate. Screening the MMV Box elucidated additionally two entirely novel chemical scaffolds. The PK trials emphasized strong impacts of an adult Schistosoma infection on PK parameters and underlined the need for further studies in that field. Finally interesting parameters for the antischistosomal drug-screening cascade were elucidated in the course of this thesis

Additional file 5: Figure S3. of Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays

Fluorescence generated from culture medium as measured by (A) Omnicathepsin, (B) DAPI and (C) Hoechst 33258. Since our standard culture medium could also contribute to high fluorescence, RPMI medium was also tested. Graphs presented here correspond to optimal marker concentrations and incubation times are indicated in the main text. (PPTX 79 kb

Boletín de Segovia: Número 131 - 1838 noviembre 8

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

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

Worm burden reductions observed for the five lead candidates in <i>S. mansoni</i> infected mice.

<p>Mice harbored a patent <i>S. mansoni</i> infection. Different dosage regimens were used (1×400 mg/kg, 4×80 mg/kg on four consecutive days or 4×100 mg/kg every 4 hours).</p><p>WBR: Worm burden reduction.</p><p>p-value<0.05.</p><p>p-value<0.005.</p

Chemical structures of the five lead compounds selected for <i>in vivo</i> studies.

<p>Chemical structures of the five lead compounds selected for <i>in vivo</i> studies.</p

Adult worm IC₅₀ values of five <i>in vivo</i> candidates over time post drug exposure.

<p>Values were determined 1, 2, 4, 7, 10, 24, 48 and 72>33.3 µM are indicated with (<b>//</b>). PZQ: praziquantel.</p

Screening flow.

<p>Screening was conducted at the Swiss TPH (screening cascade <b>I</b>; steps <b>A</b>–<b>E</b> and Quality control): Primary screening steps (yes/no filters) of 100 µM and 33.3 µM resulted in 179 and 72 hits, respectively. Active compounds (n = 72) moved on to Step <b>C</b> and IC₅₀ values were evaluated on NTS. Thirty-four compounds showed activities with IC₅₀ values <10 µM and pre-screening was conducted on adult schistosomes (Step <b>D</b>). Active compounds (n = 16) with schistosomicidal effects at 33 µM compound concentration were further characterized (step <b>E</b>). The quality control represents randomly selected compounds from compounds classified as non-active from the pre-screening steps (step <b>A</b>/<b>B</b>) on NTS which were re-evaluated in step <b>B</b>. In parallel, all compounds (n = 400) were studied at the LSHTM in London (screening cascade <b>II</b>): step <b>A</b>, all 400 compounds were screened on <i>S. mansoni</i> adults at 15 µM. Step <b>B: 44</b> compounds were active and these were then tested for IC₅₀ determination on adult worms. From both screening cascades, five compounds were selected for <i>in vivo</i> testing based on pharmacodynamic and pharmacokinetic properties as well as toxicity.</p