The MMV Box [24], containing 400 compounds as stock solutions dissolved in dimethylsulfoxide (DMSO), concentration 10 mM, was kindly provided by MMV/SCYNEXIS, Inc. (Geneva, Switzerland; Durham, USA). For the in vitro studies on adult worms at the Swiss TPH and the in vivo studies in mice 5–100 mg of 1: MMV000963, 2: MMV665852, 3: MMV665807, 4: MMV019555, 5: MMV019918, 6: MMV000445, 7: MMV019780, 8: MMV665927, 9: MMV665941, 10: MMV000634, 11: MMV665830, 12: MMV666054, 13: MMV009063, 14: MMV007591, 15: MMV665969, 16: MMV666070, 17: MMV007224, 18: MMV665794, 19: MMV666057, and 20: MMV665799 were purchased from Specs (Delft, Netherlands), and MolPort (Riga, Latvia). Praziquantel was purchased from Sigma-Aldrich (Buchs, Switzerland) GmbH. Compounds 1–20 were dissolved in DMSO for drug stock solutions of 10 mg/ml for in vitro evaluations. Culture medium for newly transformed schistosomula (NTS) was made by supplementing Medium 199 (Lubioscience, Lucerne, Switzerland) with 5% heat-inactivated fetal calf serum (iFCS), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Lubioscience, Lucerne, Switzerland). Culture medium for adult worms was prepared by supplementing RPMI 1640 with 5% iFCS, penicillin (100 U/ml), and streptomycin (100 µg/ml).

S. mansoni cercariae (Liberian strain) were harvested from infected intermediate host snails (Biomphalaria glabrata) following in-house standard procedures. Collected cercariae were mechanically transformed to NTS as described previously [25], [26]. The obtained NTS suspension was adjusted to a concentration of 100 NTS per 50 µl using supplemented Medium 199. NTS suspensions were incubated (37°C, 5% CO₂ in ambient air) for a minimum of 12 to 24 hours until usage to ensure completed conversion into schistosomula [27].

In vivo studies were conducted at the Swiss TPH, Basel, and approved by the veterinary authorities of the Canton Basel-Stadt (permit no. 2070) based on Swiss cantonal and national regulations. Experimentation at LSHTM was carried out under the UK Animals Scientific Procedures Act 1986 with approval from the LSHTM Ethics committee.

Animals (female NMRI, 3-week old, weight ca. 14 g) were purchased from Charles River (Sulzfeld, Germany) and allowed to adapt under controlled conditions (temperature ca. 22°C; humidity ca. 50%; 12-hour light and dark cycle; free access to rodent diet and water) for one week. Mice were infected by subcutaneous injection with ∼100 S. mansoni cercariae each, harvested from infected snails. For in vitro studies on adult flukes, schistosomes were collected from the hepatic portal and mesenteric veins of infected mice 7–8 weeks post infection [28]. Freshly harvested schistosomes were placed in supplemented RPMI culture medium, quickly rinsed, and stored at 37°C, 5% CO₂ until usage.

Initially, all compounds were tested at a concentration of 100 µM on S. mansoni NTS. Active compounds progressed into a secondary screening at 33.3 µM. For this purpose drug stock solutions were diluted in 96-flat bottom well plates (BD Falcon, USA) with supplemented Medium 199 and 50 µl of prepared NTS suspension (100 NTS/well) to the desired final concentration of 100 µM or 33.3 µM, respectively. Each drug was tested at least in triplicate and the highest concentration of DMSO served as control. Plates were incubated at 37°C, 5% CO₂. NTS were evaluated by microscopic readout (Carl Zeiss, Germany, magnification 80–120×) using a viability scale as previously described with regard to death, changes in motility, viability, and morphological alterations 72 hours post drug exposure [25], [26]. To ensure the accuracy of our assay, 45 compounds that lacked activity at one of the tested concentrations, were randomly selected and retested at 33.3 µM. Compounds that killed the NTS at 72 hours after exposure in at least one well were deemed active and selected for further testing.

In the next step, the IC₅₀ was determined for active compounds from the preceding screens. Drug dilution series were prepared in 96-flat bottom well plates with concentrations 2.1, 4.2, 8.4, 16.7, and 33.3 µM using supplemented culture medium. The prepared NTS suspension was then added to each well and plates were incubated at 37°C, 5% CO₂. NTS incubated in the presence of the highest DMSO concentration and praziquantel served as control. Drug effects on NTS were evaluated 72 hours post exposure, using a viability scale, as described above. Each concentration was tested in duplicate and experiments were repeated once.

Compounds presenting IC₅₀ values ≤10 µM were then tested at a concentration of 33.3 µM on adult worms in duplicate. Drug stock solutions (10 mM) were diluted in supplemented RPMI 1640 culture medium reaching a final concentration of 33.3 µM in 24-flat bottom well plates (BD Falcon, USA) within a final volume of 2.4 ml. At least three schistosomes of both sexes were added to each well. Schistosomes incubated in the presence of the highest concentration of DMSO served as control. Plates were incubated for 72 hours at 37°C, 5% CO₂. Seventy-two hours post drug exposure S. mansoni were examined phenotypically by microscope using the motility scale described before [29]. Drugs leading to the death of schistosomes 72 hours post exposure were characterized further and their IC₅₀ (IC₉₀) values were determined. Specifically, drug dilution series were prepared in 24-flat bottom well plates (BD Falcon, USA) with concentrations of 0.31, 0.93, 2.78, 8.33, and 25.0 µg/ml using supplemented RPMI culture medium and freshly prepared drug stock solutions (10 mg/ml). At least three schistosomes of both sexes were added to each well and plates were incubated at 37°C, 5% CO₂. Parasites incubated in the highest DMSO concentration and praziquantel served as controls. Drug effects were evaluated 72 hours post exposure as described above. Each concentration was tested in duplicate and trials were repeated once.

Adult worm drug testing was performed as previously reported [29] with some modifications as described. Worms of a Puerto Rican strain of S. mansoni were obtained by portal perfusion of CD1 mice (Charles River, UK) 6 weeks post-infection. Three pairs of worms were added to the wells of 48-well plates (Nunc, UK) in 1 ml complete DMEM medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (cDMEM). Compounds were tested at 15 µM containing 0.15% DMSO in single wells. Negative controls contained worms cultured in cDMEM alone and in cDMEM with 0.15% DMSO. Positive control wells contained worms cultured in praziquantel (Sigma-Aldrich, UK) at 10 µM. Cultures were incubated at 37°C and 5% CO₂. Effects were assessed on day 5 of culture using an inverted microscope (Leitz Diavert Wetzlar, Germany). Any compounds producing complete immotility or ≥70% worm motility inhibition plus severe morphological damage were considered hits in the primary screen [29]. Active compounds were then tested for IC₅₀ value determination at a concentration range from 0.55–15 µM in single wells.

The onset of action (length of time needed before an antischistosomal effect was visible) was determined for selected compounds in vitro by evaluating the IC50 at a time-range of 1–72 hours (1, 2, 4, 7, 10, 24, 48, and 72 hours) post drug exposure, as described above. The onset of action of praziquantel was also studied. Additionally, possible protein binding effects were studied for three lead candidates and praziquantel. For that purpose RPMI medium was supplemented with two different iFCS concentrations (0% and 50%) and IC50 values were calculated for the different conditions. Furthermore, IC50 values were determined after varying drug exposure times (1, 2, or 4 hours) followed by incubation in drug free RPMI medium for 72 hours.

Groups of 3–4 NMRI mice characterized by a patent S. mansoni infection (49 days post-infection) were treated orally with the test drug using either single oral doses of 400 mg/kg or 80 mg/kg administered on four consecutive days. An additional dosage regimen of 100 mg/kg administered four times every 4 hours was tested for the 2 most active compounds (2, 17). Compounds were freshly prepared in an aqueous hydroxypropyl methyl cellulose (HPMC) (1%): DMSO (95∶5) formulation. Eight to sixteen untreated mice served as controls. Fourteen days post-treatment animals were killed by the CO₂ method and were dissected and the worms were sexed and counted [28]. Mean worm burdens of treated mice were compared to the mean worm burden of untreated animals and worm burden reductions were calculated.

Parasite viability values of NTS and adult schistosomes obtained from microscopic evaluation were averaged (means (+/− standard deviation)) using Microsoft Excel. IC50 and IC90 values of test compounds were determined using the CompuSyn software (Version 3.0.1, 2007; ComboSyn Inc., USA) and Microsoft XLfit version 5.1.0.0 (2006–2008 ID Business Solutions Ltd). Selectivity indices were calculated by dividing the IC50 of the MRC-5 cells-fibroblast cytotoxicity data by the IC50 of the adult worm assay. The Kruskal-Wallis test was applied for in vivo studies, comparing the worm burden of the treated animals and control animal groups. A difference in worm burden was considered to be significant at a significance level of 5% (StatsDirect, version 2.7.2.; StatsDirect Ltd., UK).

Exposing schistosomula to the test drugs (n = 400) at a concentration of 100 µM resulted in death of NTS for 45% of the tested compounds (n = 179). Schistosomicidal effects were observed for 18% of these active compounds (n = 72) at the lower concentration of 33.3 µM (Figure 1). A diverse range of chemical scaffolds was observed amongst active compounds. Successful candidates were characterized further on NTS. Promising antischistosomal activity (IC₅₀: 1.4–9.5 µM) was observed for 34 compounds, two of which were identified during our quality control re-evaluation of 45 compounds and nine of which showed comparable or increased activity (IC₅₀: 1.4–2.4 µM) to praziquantel (IC₅₀: 2.2 µM).

All hits (IC₅₀<10 µM) (n = 34) were next tested at a concentration of 33.3 µM on adult S. mansoni. Seventy-two hours post drug exposure, 16 (1–16) of these compounds (Table S1) killed the adult worms. Four of the ten compounds with high activities (IC₅₀<2.5 µM) on NTS lacked antischistosomal activity on adult worms. The 16 active candidates were further characterized by IC₅₀ value determination. The highest in vitro activities were observed for the diaminoquinazoline derivative 1 (IC₅₀: 0.8 µM) the diarylurea 2 and diarylamide 3, presenting IC₅₀ values of 0.8 and 1.3 µM, respectively (PZQ: 0.2 µM). IC₅₀ values ranging from 2.6–9.2 µM were calculated for compounds 4–11, whereas only moderate activity (IC₅₀ values >10 µM) was determined for five compounds (12–16). Compounds with IC₅₀>10 µM were excluded from further consideration, meaning only eleven compounds were considered as hits.

Forty-four compounds were classified as hits (compounds producing complete immotility or ≥70% worm motility inhibition plus severe morphological damage) against adult S. mansoni in vitro at a concentration of 15 µM. These compounds were further tested for IC₅₀ values (Table S1). Twelve compounds showed IC₅₀ values >15 µM. Fourteen compounds revealed IC₅₀ values between 10–15 µM. Eighteen compounds had IC₅₀ values <10 µM. To provide a comparison with the Swiss TPH assays, the 32 hits were subsequently tested using the schistosomula assay at LSHTM [30]. This showed generally good concordance with the LSHTM adult assay, in that all adult hits with IC₅₀<10 µM were also hits in the larval assay (Table S1).

Based on in vitro performance on the adult worms (Table S1), toxicity, pharmacokinetic (PK) properties and availability of the compounds, five lead candidates (1, 2, 5, 8, 17) (Figure 2) were selected for in vivo testing and in depth characterization in vitro. In more detail, 11 compounds were excluded after comparing their IC₅₀ values and PK parameters (C_max, t_max, t_1/2, AUC). Four compounds showed poor antischistosomal activity (IC₅₀>10 µM) and four compounds showed poor bioavailability (C_max<IC₅₀ of the corresponding compound). Ten compounds were characterized by low selectivity indices (SI<1) and two were not commercially available.

Four active compounds were derivatives belonging to the class of diarylureas and two compounds were characterized as dianilinoquinoxalines. Only the most active candidate of each chemical group, compound 2 and compound 17, was selected for in vivo studies. A summary of the IC₅₀ values, toxicity and pharmacokinetic parameters of the lead candidates is provided in Table 1.

The onset of action was studied in compounds selected for in vivo testing (n = 5) and compared to the onset of action for praziquantel (Figure 3). Compound 2 was the fastest acting drug, presenting an IC₅₀<5 µM already after 1 hour of in vitro exposure, followed by compound 17 with an IC₅₀<10 µM, 1 hour post incubation. Compound 1 was intermediate in speed with an onset time of 7 hours post-incubation. Compound 8 had fully exerted its antischistosomal properties 24 hours following incubation, while compound 5 was slow acting (exposing its full antischistosomal activity only 72 hours post treatment). In comparison, praziquantel exposed its entire antischistosomal activity already after 1 hour of drug exposure (IC₅₀: 0.2 µM).

The determined IC₉₀ values of the lead candidates were 2–5 fold higher than the observed IC₅₀ values 72 hours post exposure and thus the concentration-response curves for these compounds are quite steep (Table 2). Comparatively, praziquantel even showed a 13-fold difference between the two values. Praziquantel lead very quickly to a strong motility inhibition and morphological changes, whereas higher concentrations (IC₉₀: 2.0 µM) were necessary to actually kill the worms.

Compound 2 and 17 revealed the highest in vivo activity with worm burden reductions (WBR) of 52.5% (dosage 1×400 mg/kg; p<0.005) and 53.4% (dosage 4×100 mg/kg; p<0.005), respectively (Table 3). In addition, both treatment regimens using multiple doses of compound 2 resulted in significant worm burden reductions of 46.0% (4×80 mg/kg; p<0.005) and 31.2% (4×100 mg/kg; p<0.05). Treatment with a single 400 mg/kg dose of compound 17 resulted in a significant worm burden reduction of 40.8% (p<0.05), while multiple treatment courses of 80 mg/kg over four consecutive days achieved a lower effect (WBR: 25.5%, p<0.05). Compounds 1, 5, and 8 lacked in vivo activity (WBR 0–18.7%). No significant differences were observed between total and female worm burden reductions.

Compound 17 showed a 7-fold increase in activity in iFCS-free medium (IC₅₀: 0.3 µM) versus incubation in 50% serum supplemented medium (IC₅₀: 2.1 µM) (Table S2). A strong increase in activity in serum free medium was observed for praziquantel (IC₅₀: 0.02 µM). No altered activities were detected for compound 2 within varying iFCS-concentrations. Short-term exposure of schistosomes to compound 2 or praziquantel (1–4 hours) followed by incubation in drug free medium for 72 hours resulted in high IC₅₀ values, ranging from 51.1 µM (1 hour) to 24.6 µM (4 hours) for compound 2 and from 96.1 µM to 7.7 µM for praziquantel (Figure S3). These values are much higher than the IC₅₀ values determined when the worms are continuously exposed to the drugs for 72 hours (2: IC₅₀: 0.8 µM; PZQ: IC₅₀: 0.2 µM). Incubation of schistosomes for 4 hours with compound 17 achieved similar effects (IC₅₀: 1.3 µM) (Figure S3) as described for the 72 hours exposure time (IC₅₀: 0.8 µM) (Table S2).