Screening open access compound libraries and repurposing drugs to identify and characterize new molecules active against schistosomiasis and other helminthiases

Far from the baneful impact of the so-called “big three” (HIV/AIDS, tuberculosis and malaria), schistosomiasis and other worm infections, such as the soil-transmitted helminths (STH) for example, have a backstairs influence on poverty in the tropical belt, mainly in sub-Saharan Africa and South-East Asia. The lack of sanitary infrastructure in endemic areas is contributing to the transmission of the parasites and the maintenance of their life-cycles in the environment. People, especially children, get infected while carrying out their daily activities or simply by walking barefoot. Preventive chemotherapy is the most widely used control strategy for schistosomiasis and STH. It relies on praziquantel for schistosomiasis and on the benzimidazoles for STH. A corollary of this repeated and large-scale use of the same drugs is that the risk of resistance emergence is rising and neither alternative treatment nor vaccine are available against these diseases. Also, the current anthelminthic formulations are not optimized for all patient categories like pregnant women or pre-school-age children. Unfortunately, because of the lack of return on investment, the industry had turned its back on anthelminthic research and development and the academic research in this area is scarce and generally underfunded. Hence, there is a dramatic lack of molecules in the development pipeline, especially in pre-clinical or clinical phases. It is a crucial moment to intensify research on antischistosomal drugs. However, it remains the appanage of only a few academic institutions throughout the world that use different screening methods and that often have a long-standing know-how on life-cycle maintenance. In order to promote early antischistosomal drug discovery and help researchers starting to work in this area, we developed a protocol unifying cultivation methods with in vitro and in vivo drug testing. In this context, the main objective of this thesis was to expand the pool of antischistosomal candidates by screening two libraries of 400 compounds each, both compiled and provided by the non-profit product development partnership Medicines for Malaria Venture (MMV). One library, the Pathogen Box, was composed of drug-like molecules with already-known activity against one or more infectious agents. The other library, the Stasis Box, was a set of drugs that were abandoned at advanced stages of their clinical development. The activity of the compounds from the two libraries was first screened in vitro on the larval stage of Schistosoma mansoni. The hits were then tested on adult worms. In both cases, the viability of the parasite was assessed phenotypically. This straightforward approach enabled the identification of 22 antischistosomal leads with satisfying in vitro activity (IC50 < 10 μM) and selectivity. However, the good efficacy in vitro did not translate in vivo, as the 16 molecules that were tested in mice harboring an infection with S. mansoni failed to significantly reduce the burden of infection. Whereas this lack of efficacy in vivo might be imputable to a strong albumin-binding effect for the Stasis Box drugs, this was not the case for the Pathogen Box compounds. In addition to test the activity of both compound libraries, a series of more than a hundred analogues from three of the leads identified in the Pathogen Box screening was also tested in vitro. Screening this set of analogues enabled to launch a preliminary structure-action relationship analysis that paves the bases for future compound synthesis programs and selected, new leads. Hundreds of Pathogen Boxes were distributed to research teams across the globe, including some that tested its compounds on schistosomes. The variability of results between laboratories screening the same set of compounds might be important, especially since there is no consensus on drug screening methods for schistosomes. Also, the reliability of activity-based phenotypic screening approach can be limited. For these reasons, our results from the Pathogen Box larval stage screening were compared to the ones obtained at the University of California in San Diego (UCSD) and the ones from metabolic assays performed by the Fiocruz Foundation in Brazil. This resulted in a 74% overall agreement between the three laboratories and confirmed that activity-based phenotypic assays on the larval stage are a reliable and cost-effective method to screen large compound libraries. In order to potentially improve the quality and increase the speed of the larval assays read-outs, we tested an image-based motility assessment method in collaboration with the team of Prof. Britta Lundström-Stadelmann at the University of Bern. Good correlations with the phenotypic assessment were found but this system must be validated with more drugs and under different conditions. Additionally, in the framework of the Master thesis of Tanja Karpstein, the screening work on schistosomes was extended to other parasites. The veterinary anthelminthic emodepside was tested not only on schistosomes but also on five different species of nematodes, including hookworms and the whipworm. While its efficacy on S. mansoni and S. haematobium remained moderate, emodepside revealed very promising in vitro and in vivo activity that should encourage its repositioning as treatment for human soil-transmitted helminthiases. In conclusion, this work describes, applies and explores different methods in anthelminthic early drug development with a strong emphasis on schistosomiasis. Activity-based screening of open access libraries has identified new potent antischistosomal molecules. Investigating the activity of some lead analogues has also enabled to single out different patterns that could be used for further compound development. Finally, this work highlights the importance of open access libraries, drug repurposing and non-profit product development partnerships to stimulate the development of new anthelminthic drugs

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery

Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel.; Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD; +; /NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis.; For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline.; The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified

Screening a repurposing library, the Medicines for Malaria Venture Stasis Box, against Schistosoma mansoni

Abstract Background The development of new treatments against schistosomiasis is imperative but lacks commercial interest. Drug repurposing represents a suitable strategy to identify potential treatments, which have already unblocked several essential steps along the drug development path, hence reducing costs and timelines. Promoting this approach, the Medicines for Malaria Venture (MMV) recently distributed a drug repurposing library of 400 advanced lead candidates (Stasis Box). Methods All 400 compounds were initially tested in vitro against the larval stage of Schistosoma mansoni at 10 μM. Hits progressed to screening on adult worms and were further characterised for IC50, cytotoxicity and selectivity. Ten lead compounds were tested in mice harbouring a chronic S. mansoni infection. Results Eleven of the 37 compounds active on the larval stage were also highly active on adult worms in vitro (IC50 = 2.0–7.5 μM). IC50 values on adult S. mansoni decreased substantially in the presence of albumin (7.5–123.5 μM). Toxicity to L6 and MRC cells was moderate. A moderate worm burden reduction of 51.6% was observed for MMV690534, while the other 9 compounds showed low activity. None of the in vivo results were statistically significant (P > 0.05). Conclusions Phenotypic screening of advanced lead compounds is a simple and resource-low method to identify novel anthelminthics. None of the promising hits of the Stasis Box identified in vitro against S. mansoni yielded acceptable worm burden reductions in vivo, which might be due to the high plasma protein binding. Since the in vitro hits interfere with different drug targets, they might provide a starting point for target based screening and structure-activity relationship studies

Early antischistosomal leads identified from in vitro and in vivo screening of the medicines for malaria venture pathogen box

As part of the control and elimination strategy of human schistosomiasis, preventive chemotherapy relies on a single drug, praziquantel. Facing an almost dry drug development pipeline, screening the Pathogen Box from the Medicines for Malaria Venture (MMV), provides a unique opportunity to possibly expand the pool of potent molecules against schistosomiasis. The activity of 400 compounds from this open-access library was first screened in vitro on the larval stage of Schistosoma mansoni. The hits were then tested on adult worms. Eleven leads were identified and tested for albumin-binding and activity on adult S. haematobium. In parallel, a rudimental structure-activity relationship analysis was performed on the 112 available analogues of three leads, yielding another 30 molecules active against both larval and adult stages of S. mansoni. Seven leads, selected on druglikeness, pharmacokinetic properties, and availability, plus auranofin were tested in mice harboring a chronic S. mansoni infection. MMV022029 and MMV022478 revealed the highest worm burden reductions of 67.8 and 70.7%, respectively. This study provided a series of new potent scaffolds and pharmacophores that could be used to design and develop suitable alternative(s) to praziquantel

Early Antischistosomal Leads Identified from in Vitro and in Vivo Screening of the Medicines for Malaria Venture Pathogen Box

As part of the control and elimination strategy of human schistosomiasis, preventive chemotherapy relies on a single drug, praziquantel. Facing an almost dry drug development pipeline, screening the Pathogen Box from the Medicines for Malaria Venture (MMV), provides a unique opportunity to possibly expand the pool of potent molecules against schistosomiasis. The activity of 400 compounds from this open-access library was first screened in vitro on the larval stage of Schistosoma mansoni. The hits were then tested on adult worms. Eleven leads were identified and tested for albumin-binding and activity on adult S. haematobium. In parallel, a rudimental structure-activity relationship analysis was performed on the 112 available analogues of three leads, yielding another 30 molecules active against both larval and adult stages of S. mansoni. Seven leads, selected on druglikeness, pharmacokinetic properties, and availability, plus auranofin were tested in mice harboring a chronic S. mansoni infection. MMV022029 and MMV022478 revealed the highest worm burden reductions of 67.8 and 70.7%, respectively. This study provided a series of new potent scaffolds and pharmacophores that could be used to design and develop suitable alternative(s) to praziquantel

Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni

Drug discovery for schistosomiasis is still limited to a handful of academic laboratories worldwide, with only a few novel antischistosomal lead compounds being actively researched. Despite recent international mobilization against the disease to stimulate and promote antischistosomal drug discovery, setting up a drug-screening flow with schistosome parasites remains challenging. Whereas numerous different protocols to obtain and cultivate schistosomes have been published, those describing the drug-screening process are scarce, and none gather together parasite cultivation and early drug discovery procedures. To help overcome this hurdle, we provide here a set of integrated methods either adapted from already-published protocols or based on our long-term experience in schistosomiasis research. Specifically, we detail the establishment and maintenance of the complex and several-week-long Schistosoma mansoni life cycle in a laboratory setting, as well as the means of retrieving and culturing the parasites at their relevant life stages. The in vitro and in vivo assays that are performed along the drug-screening cascade are also described. In these assays, which can be performed within 5 d, the effect of a drug is determined by phenotypic assessment of the parasites' viability and morphology, for which stage-specific scoring scales are proposed. Finally, the modalities for testing and evaluating a compound in vivo, constituting a procedure lasting up to 10 weeks, are presented in order to go from in vitro hit identification to the selection of early lead candidates

Evaluation of emodepside in laboratory models of human intestinal nematode and schistosome infections

Abstract Background Helminthiases are very prevalent worldwide, yet their treatment and control rely on a handful of drugs. Emodepside, a marketed broad-spectrum veterinary anthelminthic with a unique mechanism of action, undergoing development for onchocerciasis is an interesting anthelmintic drug candidate. We tested the in vitro and in vivo activity of emodepside on nematode species that serve as models for human soil-transmitted helminth infection as well as on schistosomes. Methods In vitro viability assays were performed over a time course of 72 hours for Trichuris muris, Necator americanus, Ancylostoma ceylanicum, Heligmosomoides polygyrus, Strongyloides ratti, Schistosoma mansoni and Schistosoma haematobium. The drug effect was determined by the survival rate for the larvae and by phenotypical scores for the adult worms. Additionally, mice infected with T. muris and hamsters harboring hookworm infection (N. americanus or A. ceylanicum) were administered orally with emodepside at doses ranging from 1.25 to 75 mg/kg. Expelled worms in the feces were counted until 3 days post-drug intake and worms residing in the intestines were collected and counted after dissection. Results After 24 hours, emodepside was very active in vitro against both larval and adult stages of the nematodes T. muris, A. ceylanicum, N. americanus, H. polygyrus and S. ratti (IC50 < 4 µM). The good in vitro activity was confirmed in vivo. Hamsters infected with the hookworms were cured when administered orally with 2.5 mg/kg of the drug. Emodepside was also highly active in vivo against T. muris (ED50 = 1.2 mg/kg). Emodepside was moderately active on schistosomula in vitro (IC50 < 8 µM) 24 h post-drug incubation and its activity on adult S. mansoni and S. haematobium was low (IC50: 30–50 µM). Conclusions Emodepside is highly active against a broad range of nematode species both in vitro and in vivo. The development of emodepside for treating soil-transmitted helminth infections should be pursued

Multi-center screening of the Pathogen Box collection for schistosomiasis drug discovery.

BACKGROUND:Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS:Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72&nbsp;h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS:For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72&nbsp;h, concordance remained a robust 74%. Further, robust&nbsp;Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72&nbsp;h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS:The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified