Parasitic worms infect more than 1.5 billion people and cause significant losses in livestock and crops. New anthelmintic drugs are urgently needed, as resistance to the existing drugs is emerging. We built a high-throughput (HTP) and high-content robotic screening platform to identify bioactive small molecules and their molecular targets, focusing on novel disease therapeutics and broad spectrum anthelmintics. The platform is capable of screening compound collections and genetic libraries in model organisms and mammalian cells in a fully integrated manner. To find novel anthelmintics we screened a ~2300 compound library containing most of the FDA-approved approved drugs, plus natural products and bioactive compounds for broad spectrum toxic effects on two distantly related free living nematodes C. elegans and P. pacificus as model animals while having low toxicity on human cell lines. The screen confirmed the effects of most known anthelmintics on these nematode models. Additionally, among a set of new compounds, we found six natural lipid molecules that exhibit broad-spectrum anthelmintic activity. These novel compounds kill C. elegans and P. pacificus when applied at various developmental stages, including the dauer and embryonic stages, in a dose-dependent manner. Importantly, these compounds cause mortality in all three veterinary parasite species we tested: the hookworm Haemonchus contortus (ruminants), Teladorsagia circumcincta (sheep and goat) and Heligmosomoides polygyrus (rodents). Further characterization of their mode of action using C. elegans revealed defects associated with mitochondrial function and lipid metabolism. The lethality phenotype in C. elegans is partially rescued by knocking down the enzymes
acs-2,
cpt-4,
dif-1 and
cpt-2, which facilitates mitochondrial lipid transfer, while drug inhibitors of CPT1 and CPT2 enhance the phenotype, suggesting that these compounds perturb specifically fatty acid oxidation in nematodes. These compounds constitute a new class of broad-spectrum anthelmintics targeting lipid metabolism.