The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets

New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target

Escape from larval arrest on IVM.

<p>Worms with AVR-15::YFP expressed under control of <i>acc-2</i>, <i>acc-3</i>, <i>lgc-47</i>, and <i>lgc-49</i>-promoters fail to develop on concentrations of IVM above 200ng/mL. Percent survival was normalized to the plates with the highest total number of L1-escaped worms (either 0.1 or 10ng/mL IVM), n = 4 plates of worms per concentration, error bars ± 1 SEM.</p

Deletions in various <i>acc</i> genes result in subtle phenotypes.

<p><b>(A)</b> Worms with deletions in <i>acc-1</i> (tm3268) but not <i>acc-2</i> (ok2216) have slightly slowed pharyngeal pumping rates; error bars ± 1SEM, * indicates p<0.05. <b>(B)</b> Worms with deletions in <i>acc-1</i> (tm3268) but not <i>lgc-47</i> (ok2963) have decreased egg-laying; error bars ± 1SEM, ** indicates p<0.001. <b>(C, D)</b> Worms with deletions in <i>lgc-49</i> (gk246966) and <i>acc-2</i> (ok2216) have slightly slowed development when compared to wildtype N2.</p

Schematic representation of our strategy to validate the ACCs as targets of agonist drugs.

<p>Wild-type <i>C</i>. <i>elegans</i> express the IVM-targeted GluCls in essential tissues and thus are sensitive to IVM. The strain JD369 lacks functional IVM-targeted GluCl channels, and is thus insensitive to IVM. By selectively reintroducing the GluCl channel subunit AVR-15 under the control of the different ACC promoters, we are able to generate strains that have IVM-gated channels exclusively in tissues that endogenously express the ACC channels. We can then treat these strains with IVM to predict the effects of a direct ACC agonist. Green neurons represent endogenous GluCl-expressing neurons. Red neurons indicate ACC-expressing neurons. Grey neurons indicate loss of GluCl expression in endogenous cells. Yellow neurons indicate GluCl AVR-15::YFP transgene expressed in ACC-expressing cells.</p

<i>Pacc</i>::AVR-15::YFP constructs are expressed in neural tissues throughout the worm.

<p>All images show representative epifluorescence of tagged YFP-tagged AVR-15 channels expressed under control of ACC promoters. <b>(A)</b><i>Plgc-48</i>::AVR-15::YFP expression in non-essential amphid neurons in the head. <b>(B)</b><i>Plgc-48</i>::AVR-15::YFP expression in non-essential amphid neurons in the tail. <b>(C)</b><i>Plgc-47</i>::AVR-15::YFP expression. <b>(D)</b><i>Pacc-1</i>::AVR-15::YFP expression. <b>(E)</b><i>Pacc-2</i>::AVR-15::YFP expression. <b>(F)</b><i>Pacc-3</i>::AVR-15::YFP expression. <b>(G)</b><i>Plgc-49</i>::AVR-15::YFP expression. For all boxes, arrows indicate expression in ventral nerve cord, scale bars indicate 50μm.</p

Phylogenetic tree of ACCs in <i>C</i>. <i>elegans</i> and parasitic nematode species.

<p>A maximum likelihood tree made from predicted protein sequences identified using <i>C</i>. <i>elegans</i> (cel) ACCs as BLAST queries for similar sequences in <i>Haemonchus contortus</i> (Hco), <i>Ancylostoma ceylanicum</i> (Ace), <i>Necator americanus</i> (Nam), <i>Strongyloides ratti</i> (Sra), <i>Brugia malayi</i> (Bma), <i>Wuchereria bancrofti</i> (Wba), <i>Loa loa</i> (Llo), <i>Ascaris suum</i> (Asu), <i>Trichuris suis</i> (Tsu), <i>Trichuris trichiura</i> (Ttr), and <i>Trichinella spiralis</i> (Tsp). Bootstrap values out of 100 are indicated at ACC clade-defining branches. The tree was rooted to the α1 subunit of the torpedo nicotinic AChR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138804#pone.0138804.ref081" target="_blank">81</a>]. Representative subunits from other <i>C</i>. <i>elegans</i> chloride-selective pLGIC clades (LGC-50, LGC-51, LGC-52, MOD-1, EXP-1, AVR-15, GAB-1, and GGR-2) were also included to ensure that predicted ACC orthologs from other species grouped with the celACCs instead of other <i>C</i>. <i>elegans</i> chloride channels. Inset: Phylogenetic relationship of nematodes species. Branch lengths are approximate.</p

ACC orthologs in parasitic nematodes.

<p>The <i>C</i>. <i>elegans</i> ACC genes are represented as columns; a value in the corresponding cell indicates the presence of an ortholog of that gene in a specific species, with the percent homology indicated. Merged cells indicate that the parasitic gene is an ortholog of an ancestral gene that gave rise to multiple ACCs in <i>C</i>. <i>elegans;</i> the highest corresponding percent homology is indicated in the table.</p><p>ACC orthologs in parasitic nematodes.</p

Adulticidal IVM activity.

<p>Well-fed young adults were placed on 500ng/mL IVM and were checked for live worms every day up to five days. Survival was assessed as described in Wilkinson et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138804#pone.0138804.ref038" target="_blank">38</a>]. Graph shows adult survival after 5 days on 500ng/mL IVM. Strains with <i>Plgc</i>-47::AVR-15 were unable to survive to day five, while all other strains, except <i>Plgc</i>-48::AVR-15 containing strains, showed reduced survival, n = 3 plates per genotype, error bars ± 1 SEM.</p

Adult paralysis on IVM.

<p><b>(A)</b> Well-fed young adults were placed on 500ng/mL IVM and were monitored every hour up to four hours for their ability to move in response to a plate tap or stimulation with a worm pick (harsh touch). All strains, except <i>Plgc</i>-48::AVR-15 containing strains, showed paralysis after four hours on 500ng/mL IVM. n = 4 plates per genotype. <b>(B)</b> After 4h of 500ng/mL IVM exposure, paralyzed worms were allowed to recover overnight and worms that moved in response to a plate tap or stimulation with a worm pick (harsh touch) were scored as recovered. n = 4 plates per genotype, error bars ± 1 SEM.</p