<i>C. elegans</i> phototaxis habituation.

<p>A classic habitation pattern is observed with continuous scanning with a 90-second interval for 20 intervals. Temperature on the scanner surfaces was monitored and found to be 20°C. The light response was measured in triplicate with 30 worms/plate.</p

Processing and analysis of scanned images.

<p>(a) A 16-bit grey scale scan of a 5.5 cm petri dish containing N2 on NGM with OP50. Exposed to 200 ppm of phosphine for 24 hours with 48 hours of recovery. Only 4.5 by 4 mm crop is shown of the 5.5 cm. The black bar represents 1 mm, or 2400 dpi. (b) The sequential scan is taken 90 seconds after first. (c) Image segmentation with particle analysis, this distinguishes the nematodes from the background. Which are outlined with red lines, labeled W1–4. (d) Image difference of consecutive scans that has been thresholded with hysteresis. Where the worm positions are overlaid. This allows for calculation of both worm movement. Mortality is defined as less than less than 10% movement. Worm positions shown in red lines and labels commenting on level of movement.</p

Using <i>C</i>. <i>elegans</i> Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

<div><p>Background</p><p>The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode <i>Caenorhabditis elegans</i> as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner.</p><p>Methods/Principal findings</p><p>Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of <i>C</i>. <i>elegans</i> when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of <i>C</i>. <i>elegans</i>. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells.</p><p>Conclusions/Significance</p><p>The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. <i>C</i>. <i>elegans</i> as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.</p></div

Mapping compound mode of action using <i>C</i>. <i>elegans</i> anthelmintic resistant strains.

<p>The IC₅₀ of different <i>C</i>. <i>elegans</i> anthelmintic resistant strains after 5 days exposure, 2 L4 stage <i>C</i>. <i>elegans</i> were placed in a 96-well for three biological replicates.</p

A structure-activity relationship (SAR) analysis of CID 2747322.

<p>(A) <i>Hierarchical maps</i>. Hierarchical maps were used to categorize 14 of the 50 compounds from the SAR screen, with variations in the right R-group 2-(trifluoromethyl)benzene, left R-group 2-(4-methoxyphenoxy)ethyl or both R-groups. Included in the hierarchical map is a Bayer patented compound (N-(2-((5-methylpyridin-2-yl)oxy)ethyl)-2-(trifluoromethyl)benzamide) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005058#pntd.0005058.ref065" target="_blank">65</a>], which has a 3D similarity score of 0.82. Fluopyram has a 3D similarity score of 0.58. Also included is the WACT-11 compound identified by Burns [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005058#pntd.0005058.ref034" target="_blank">34</a>] with a 3D similarity score of 0.66, and the flutolanil analogue (CID 49852661) used for modeling which has a 3D similarity score of 0.60. (B) <i>A SAR analysis for CID 2747322 against our internal library of 24</i>,<i>989 compounds from Maybridge and Chembridge libraries</i>. The top 50 compounds with the highest 3D similarity scores to CID 2747322, calculated using Screen3D version 2015 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005058#pntd.0005058.ref057" target="_blank">57</a>] were re-pinned for two additional biological replicates. Compounds with variations on the left R-group 2-(trifluoromethyl)benzene or the right R-group 2-(4-methoxyphenoxy)ethyl or both R-groups are highlighted. The compound CID 2747279 (3D Similarity Score of 0.74), has variations on the right R-group was the only compound from the set of 50 that displayed strong anthelmintic activity.</p

A phylogenetic comparison of the quinone binding site in MEV-1.

<p>Sequences for <i>mev-1</i> of representative nematode species together with, <i>Homo sapiens</i>, <i>Sus scrofa</i>, and <i>S</i>. <i>cerevisiae</i> were obtained using BlastP and aligned by ClustalW. The key residues involved in quinone binding, are conserved amongst all nematode species examined. The red star indicates the T66I variant that results in resistance to CID 2747322 exposure in the VC3631 strain. Residues important for the left binding pocket of CID 2747322 are indicated with a black star. Image was generated using Geneious version 8.1.7.</p

Multiple mitochondrial complex II mutations confer resistance to CID 2747322.

<p>To confirm that mev-1 (<i>gk361</i>) is the cause of resistance in strain VC3631 to CID 2747322 we tested mitochondrial complex II mutant lines from the <i>C</i>. <i>elegans</i> Million Mutation Project (MMP). It was found that 7 of the MMP strains for the mitochondrial complex II were resistant towards CID 2747322 exposure, 15 of the strains had wild-type (none) resistance and 9 strains were sensitive towards exposure.</p

WormScan Scores from initial compound screen and confirmation of hits.

<p>(A) Two VC2010 L4 <i>C</i>. <i>elegans</i> were sorted into each well of a 96-well flat-bottom plate. <i>C</i>. <i>elegans</i> were exposed to 43 μM for each of the 26,000 compounds from the Maybridge and Chembridge libraries for 5 days and then screened and sorted by WormScan Score, which was normalized by percent of control wells. The 404 top anthelmintic candidates from the initial screen are highlighted by the black box. (B) The WormScan Scores of the 5,152 controls from the compound screen are shown in histogram with a bin width of 2. The mean WormScan Score for the controls is 100. (C) The top 404 compounds were re-pinned for two more biological replicates and displayed here are the top 184 active compounds. After a series of filters were applied to these 184 compounds, 14 compound candidates were retained for further testing.</p

Copy number mtDNA of PINK-1 MMP strains.

<p>The MMP strains were previously sequenced to a coverage of ≥15x. The relative mtDNA copy number was calculated by assuming that the chromosomes have two copies it is possible to scale the number of reads with the size of the chromosome and mtDNA and get an estimate of the copy number of mtDNA.</p

WormScan Score Analysis.

<p>(A) Two VC2010 L4 stage <i>C</i>. <i>elegans</i> in a 1% DMSO control well that contains no drug, two sequential scans are taken after five days of exposure, the difference image generated for the region of interest (black circle) gives a WormScan Score of 100. (B) Two VC2010 L4 <i>C</i>. <i>elegans</i> exposed to 43 μM of CID 6741218 for five days exposure resulted in reduced brood size, reduced behavioral response to light stimulus and increased mortality, giving a WormScan Score of 50. (C) Two VC2010 L4 stage <i>C</i>. <i>elegans</i> exposed to 50 μM of Ivermectin for five days of exposure, giving a WormScan Score of 0, which resulted in mortality. (D) A well that contained no <i>C</i>. <i>elegans</i> gives a WormScan Score of 0. The scale bar applies to all images.</p