In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

GO biological process enrichment for the 882 deletion strains below the threshold of detection by microarray in the BCprot relative to the gene universe of strains present in at least one deletion collectio

Average log2ratios for all conditions tested by Bar-seq from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

For Bar-Seq data generated for each deletion collection and drug or environmental experimental condition relative to its control, average log2ratios for condition replicates are given

Supplemental figure legends, additional files and references from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

Supplemental figure legends, additional files and reference

GO enrichment of deletion strains present in the HLU signature from Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress

GO biological process enrichment for the 73 deletion strains present in the HLU deletion signature

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

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 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