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.