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Parasitic nematodes constitute one of the major threats to human health, causing diseases of major socioeconomic importance worldwide. Recent estimates indicate that more than 1 billion people are infected with parasitic nematodes around the world. Current measures to combat parasitic nematode infections include anthelmintic drugs. However, heavy exposure to anthelmintics has selected populations of livestock parasitic nematodes that are no longer susceptible to the drugs, rendering several anthelmintics useless for parasitic nematode control in many areas of the world. The rapidity with which anthelmintic resistance developed in response to these drugs suggests that increasing the selective pressure on human parasitic nematodes will also rapidly generate resistant worm populations. Therefore, development of new anthelmintics is of major importance before resistance becomes widespread in human parasitic nematode populations. G-Protein Coupled Receptors (GPCRs) represent an important target for many pharmacological interventions due to their ubiquitous expression in various cell types. GPCRs contribute to numerous physiological processes, and their ligand binding sites located on cell surfaces make them accessible targets and attractive substrates in terms of druggability. In fact, ∼35 % of Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved drugs target GPCRs and their associated proteins, with over 300 additional drugs targeting GPCRs at the clinical trial stage. Nematode Chemosensory GPCRs (NemChRs) are unique to nematodes, and therefore represent ideal substrates for target-based drug discovery. Here we set out to identify NemChRs that are transcriptionally active inside the host, and to use these NemChRs in a reverse pharmacological screen to impede parasitic development. Our data identified several NemChRs, and we focused on one that was expressed in neuronal cells and exhibited the highest fold change in transcription after host activation. Next, we performed homology modelling and molecular dynamics simulations of this NemChR in order to conduct a virtual screening campaign to identify candidate drug targets which were ranked and selected for experimental testing in bioassays. Taken together, our results identify and characterize a candidate NemChR drug target, and provide a chemogenomic pipeline for identifying nematicide substrates.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.