Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class.

Interactions of anthelmintic drugs in Caenorhabditis elegans neuro-muscular ion channel mutants.

Due to the increasing development of anthelmintic resistance in nematodes worldwide, it is important to search for anthelmintic compounds with new modes of action and also to investigate the possibility to combine compounds with possible synergistic effects. There might also be the chance to take advantage of the fact that nematode populations which have developed resistance against one anthelmintic class might respond hypersusceptibly to another drug class. The aim of this study was to investigate responses of Caenorhabditis elegans populations with mutations in neuro-muscular ion channels to different anthelmintic classes. Furthermore, potential synergistic effects between two anthelmintic compounds from different classes, i.e. emodepside and tribendimidine, were studied. Although there was neither a synergistic nor an antagonistic effect between emodepside and tribendimidine, other types of interactions could be identified. The C. elegans GABAA-receptor (GABAA-R) unc-49 mutants, showing decreased emodepside susceptibility, were more susceptible to tribendimidine than wild-type C. elegans. In contrast, the reverse phenomenon - hypersusceptibility to emodepside in tribendimidine resistant acetylcholine-receptor (AChR) loss of function mutants - was not observed. Moreover, the slo-1 mutant strain (completely emodepside resistant) also showed hypersusceptibility to piperazine. Interestingly, neither the GABAA-R unc-49 mutants nor the AChR mutants showed decreased susceptibility against piperazine, although there were some studies that indicated an involvement of GABAA-R or AChR in the piperazine mode of action. In conclusion, the present study provides evidence suggesting that interactions between commercially available anthelmintic drugs with different modes of action might be a relatively common phenomenon but this has to be carefully worked out for each anthelmintic and each anthelmintic drug combination. Moreover, results obtained in C. elegans will have to be confirmed using parasitic nematodes in the future.

Decreased emodepside sensitivity in unc-49 γ-aminobutyric acid (GABA)-receptor-deficient Caenorhabditis elegans.

Emodepside, a semi-synthetic derivative of PF1022A, belongs to a new class of anthelmintic drugs, the cyclooctadepsipeptides, and shows good efficacy against macrocyclic lactone-, levamisole- or benzimidazole-resistant nematode populations. Although putative receptors for emodepside have already been discovered, its mode of action is still not fully understood. The involvement of the γ-aminobutyric acid (GABA)-receptor on the PF1022A mode of action has previously been postulated. Therefore, a possible role of the GABA-receptor, unc-49, in the mode of action of emodepside was investigated using two different Caenorhabditis elegans in vitro assays, a motility assay and a development assay. It was found that there is a clearly reduced sensitivity against emodepside of strains carrying a GABA-receptor, unc-49, loss of function mutation compared with N2 wild type C. elegans. To transfer these results from the model system to parasitic nematodes, the Toxocara canis unc-49B cDNA sequence was identified and used in a rescue experiment. The emodepside-susceptible phenotype could be fully rescued by injection of the T. canis unc-49B cDNA sequence. We believe that this is the first functional rescue of a C. elegans mutant strain with a gene from a clade III parasitic nematode. These findings, together with the earlier data on GABA-receptor binding of PF1022A, suggest that the GABA(A)-receptor UNC-49 is associated with the emodepside mode of action. However, the only partially resistant phenotype of the loss of function mutants indicates that other pathways play a more significant role.

SLO-1-channels of parasitic nematodes reconstitute locomotor behaviour and emodepside sensitivity in Caenorhabditis elegans slo-1 loss of function mutants.

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5' flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.