Screening of the Pathogen box reveals novel anti blood-feeding compounds

Soil-transmitted helminth (STH) infections such as Necator americanus infect millions globally, and are a major cause of anemia and developmental stunting in low and middle income countries. Blood-feeding hookworms in particular rely on the digestion of host erythrocytes for nutrition and therefore detoxify heme as a byproduct of their parasitism. This dependency on blood feeding and subsequent detoxification renders this pathway as a vulnerable target for therapeutic intervention, particularly as it is the cause of morbidity in those infected. Here we described the continued development and application of a high-throughput in vitro assay using the so-called rodent hookworm Nippostrongylus brasiliensis, a model that shares key traits with N. americanus including blood feeding and hemozoin-like pigment formation. We optimized a fluorescence-based screening cascade to utilise GelGreen as a cost-effective viability stain and screened 400 compounds from the MMV Pathogen Box. Multiple compounds displayed enhanced activity in the presence of blood, suggesting interference with blood-feeding or blood-feeding-induced development. Five hits were selected for further validation, and as proof-of-principle of this screening cascade, all five were well tolerated in vivo at low doses in a murine model. This study therefore demonstrates this method can be used as a tractable and biologically relevant screening approach to identify compounds active against blood-feeding nematodes. Future work can further develop such compounds into lead drug candidates, and be leveraged for comparative parasitology approaches to identify pan-anthelmintic drugs.