Nematode infections cause significant morbidity and have a devastating global economic impact. Anthelmintic development has been hampered by lack of cost-effective screening platforms due, in part, to the absence of nematode cell lines, difficulties in maintaining parasitic nematodes in laboratory, and the costs of in vivo screening. In the present study we exploited the many advantages of the C. elegans model system and developed a high-throughput screening platform to identify selective nematode monoamine receptor agonists in genetically-engineered “chimeric” C. elegans as lead compound for anthelmintic development. Previously, we and others have demonstrated that exogenous monoamines, such as 5-HT, dopamine and tyramine (TA), each paralyzed the free-living nematode, C. elegans and, where examined, parasitic nematodes. Specifically, we have heterologously expressed 5-HT and TA receptors from a variety of organisms in the motor neurons and body wall muscle of different C. elegans receptor mutants, at sites yielding robust locomotory phenotypes, including paralysis, upon agonist stimulation. This approach includes nematode-specific accessory proteins and cuticle, while maintaining the unique pharmacologies of receptors from individual parasites. Using this approach, we have identified selective receptor agonists for both the nematode and human 5-HT1 receptors. Similarly, we have expressed novel nematode monoamine-gated Cl- channels in motor neurons and body wall muscle and validated their participation in a robust monoamine-mediated paralysis at both sites. In addition, we have modified the incubation system by varying incubation conditions and genetic backgrounds to dramatically increase the permeability of the chimeric animals, allowing much less drug to be used for screening. In fact, using this approach it may be possible to modify the cuticular permeability of C. elegans to mimic individual parasites species.
Finally, we have used the C. elegans model to tentatively identify the receptor for a FMRFamide neuropeptide, PF4 (KPNFIRFamide). Previous workers have demonstrated that PF4 causes a rapid, flaccid paralysis when injected into Ascaris suum and induces a rapid, Cl--dependent hyper-polarization in denervated A. suum muscle strips, suggesting that PF4 opens a Cl- channel on nematode muscle that has the potential to cause hyper-polarization and flaccid paralysis and may be a potentially important target for anthelmintic development (Maule et al., 1995a; Maule et al., 1995b; Holden-Dye et al., 1997; Purcell et al., 2002; Reintiz et al., 2011). The C. elegans and A. suum flp-1/af26 genes encode peptides similar (or identical) to PF4, suggesting a potential conservation in both ligand and its receptor. To this end, we identified a group of sensory-mediated locomotory phenotypes in flp-1 null animals and animals over-expressing flp-1. The C. elegans genome contains a number of genes encoding putative ligand-gated Cl- channels. We reasoned that null and XS alleles of the gene encoding the putative PF4 receptor(s) would have phenotypes similar to flp-1 null and XS animals. In addition, flp-1XS phenotypes should be absent in the receptor null background. This screen identified a putative receptor meeting these criteria, LGC-50. LGC-50 is highly conserved in both free-living and parasitic nematodes. As predicted, flp-1 and lgc-50 null phenotypes were identical and over-expressing lgc-50 in the body wall muscle significantly reduced locomotion in wild type, but not flp-1 null animals. Most importantly, the direct injection of PF4 caused a rapid paralysis in animals over-expressing LGC-50 in muscle. Together, these genetic data suggest that LGC-50 is a PF4 receptor; however, PF4 had no effect on Cl- currents of Xenopus oocytes expressing LGC-50. A. suum muscle expresses an uncharacterized ligand-gated Cl- channel, with high identity to the C. elegans LGC-34, based on muscle-specific transcriptome data, so we also examined lgc-34 null animals for flp-1 phenotypes. Surprisingly, lgc-34 null and over-expression phenotypes were identical to those observed in flp-1 and lgc-50 null and over-expressing animals. Most importantly, lgc-50 over-expression phenotypes were absent in lgc-34 null animals, suggesting that LGC-50 and LGC-34 might both be subunits of a PF4-gated channel. These studies are continuing to characterize the ligand-specificity of the putative LGC-50/LGC-34 heteromeric channel.
These studies highlight the power of the C. elegans model system for target identification and anthelmintic screening. Certainly, any observations from “chimeric” C. elegans will ultimately need to be verified in the target parasite, but these results validate the utility and versatility of this “dual systems” approach, where observations from free-living nematodes are translated to the parasites. Although the present study is focused on monoamine receptors, the screening protocol and approaches that we have developed have the potential to be useful in the characterization a wide range of additional anthelmintic targets.