High-throughput phenotypic screening of Medicines for Malaria Venture's Hit Generation Library 1 identifies new nematocidal chemotypes

Parasitic helminths impose substantial health and economic burdens on humans and livestock, and widespread resistance to existing anthelmintic classes underscores the urgent need for new chemotypes with distinct mechanisms of action as part of integrated parasite control strategies. Here, we performed a large-scale phenotypic screen of the Medicines for Malaria Venture Hit Generation Library 1 (HGL1), testing 139,916 compounds (98.7% of the 141,786-compound library) against exsheathed third-stage larvae of Haemonchus contortus, with selective cross-species evaluation in Caenorhabditis elegans. Using infrared-based motility and developmental assays in 384-well format, the platform delivered excellent performance across > 360 plates (mean Z' = 0.799 ± 0.012; signal-to-background = 65.6 ± 9.8). We identified 272 primary hits (0.194%) and confirmed 110 active compounds with reproducible inhibition of larval motility and development. Of these, 39 exhibited IC₅₀ < 10 μM and 33 caused complete developmental arrest at ≤ 12.5 μM, accompanied by characteristic phenotypes such as eviscerated, curved and coiled forms. Four of the 39 compounds were non-toxic to HepG2 cells (CC₅₀ ≥ 20 μM; MC₅₀ ≥ 20 μM), and a subset displayed favourable physicochemical properties (logD < 3; polar surface area < 100 Ų; metabolic stability > 60%), with ADME (absorption, distribution, metabolism and excretion) profiling available for a prioritised subset. Integration of potency, selectivity and/or ADME data enabled the prioritisation of candidates, including 16 that met stringent criteria for medicinal-chemistry progression. These findings demonstrate that a chemically curated library originally developed for antimalarial discovery might yield potent, selective nematocidal scaffolds and support a scalable framework for repurposing discovery libraries across divergent parasite groups.