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. 2015 Jun 25;6:7485.
doi: 10.1038/ncomms8485.

Caenorhabditis Elegans Is a Useful Model for Anthelmintic Discovery

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Caenorhabditis Elegans Is a Useful Model for Anthelmintic Discovery

Andrew R Burns et al. Nat Commun. .
Free PMC article

Abstract

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery.

Figures

Figure 1
Figure 1. Molecules that kill C. elegans are enriched for those that are lethal to parasitic nematodes.
(a) Flow chart outlining the multi-organism small-molecule screening pipeline. (b) Venn diagram showing the overlap of wactive library molecules that kill C. elegans, Cooperia oncophora and H. contortus. (c) Chart showing the enrichment of molecules that kill Cooperia, H. contortus, zebrafish, and HEK cells in the set of 275 C. elegans-lethals, relative to a randomly selected set of 182 compounds.
Figure 2
Figure 2. Nematode selectivity and structural profiling of the 275 C. elegans-lethal molecules.
(a) Heat map indicating the lethality (or lack thereof) induced by each of the 275 C. elegans-lethals in two species of parasitic nematode, as well as zebrafish embryos and human embryonic kidney (HEK) cells. For each species, the number of molecules that induce lethality is indicated to the right of the heat map. The molecules segregate into three groups based on their nematode selectivity and cross-species lethality. If a genetic screen for resistant mutants was performed for a given molecule, this is indicated, as well as the outcome of the screen. (b) Network based on the structural similarity of the 275 C. elegans-lethal molecules. Nodes represent molecules, and edges connect molecules with a pairwise Tanimoto/FP2 score >0.55 (see Methods). The group to which each molecule belongs is indicated by the node fill colour, whereas the genetic screen information is indicated by the node border colour. In the legend, the number of molecules is indicated in parentheses. The 19 clusters containing three or more molecules are named C1 to C19. The wact-11 structural family (cluster C10) is magnified, and the names of each molecule in the family are indicated.
Figure 3
Figure 3. Wact-11 and wact-12 resistant mutants are cross-resistant to all nine wact-11-family members.
(a) The wact-11-family core structure and the structure of an unrelated molecule, wact-2, which was used as a negative control throughout this work. (b) Heat maps of the wact-11-family dose-response experiments with wild-type worms (N2 strain), as well as two mutant strains, RP2674 and RP2698, isolated as being resistant to wact-12 and wact-11, respectively. The dose-response experiments were carried out using a 96-well plate liquid-based assay (see Methods). White indicates that there were more than 50 worms in three out of four replicate wells. Pink indicates that there were between 12 and 50 worms in three out of four replicate wells. Red indicates that there were between 0 and 11 worms in three out of four replicate wells. In the case of ties, the higher number prevailed (for example, at a given concentration, if two wells had 55 worms, and the other two wells had 20 worms, the chemical would be scored as having more than 50 worms). The R1 and R2 groups are indicated for each wact-11-family member. Wact-2 is used here as a negative control.
Figure 4
Figure 4. Complex II residues that are mutated in the wact-11 family resistant mutants cluster near the ubiquinone-binding site (Q-site).
(a) Rendering of the crystal structure of Ascaris suum Complex II bound to the Q-site inhibitor flutolanil (PDB: 3VRB). The side chains of the 14 orthologous residues that are mutated in the wact-11-family resistant mutants are shown as opaque spheres. The atoms of the bound flutolanil molecule are shown as orange-coloured opaque spheres. (b) Close-up view of flutolanil bound at the Q-site of Complex II from Ascaris suum. The 12 residues shown are no more than 4 Å away from flutolanil, and make up the flutolanil binding pocket. Intermolecular distances are indicated with bidirectional arrows. The dashed line represents a hydrogen bond (H-bond) interaction. Only those H-bonds that occur between Complex II residues and flutolanil are shown; H-bonds that occur between residues of Complex II were omitted for clarity. Bound cofactors, and a bound fumarate molecule, were also omitted for clarity.

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