The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear. We performed locomotor activity assays, and we found 5-HT and Tryp rapidly decrease worm motility, which is reversible only at low 5-HT concentrations. Mutants lacking MOD-1 are partially resistant to both drugs, demonstrating its role in locomotion. Acting as an antagonist of MOD-1, we showed PZE reduces the locomotor effects of exogenous 5-HT. Therefore, Tryp- and PZE-derived compounds, acting at MOD-1 through different molecular mechanisms, emerge as promising anthelmintic agents. This study enhances our knowledge of the function and drug selectivity of Cys-loop receptors and postulates MOD-1 as a potential target for anthelmintic therapy.
Methods
Locomotion assays. Motility assays were performed with WMicroTracker. All behavioral assays were done at room temperature (21–23ºC) with young adult hermaphrodite worms from synchronized plates. Prior to the experiment, worms were transferred from Nematode Growth Medium agar plates into a 15ml conical tube containing water, allowed to sink to the bottom and washed three times with water. Then, animals were transferred to flat bottomed 96-well microplates at an average of 50 worms per well in water. The experiments were carried out in water since less 5-HT is required than in salt-containing media.Worms’basal movement was measured for 30 min to normalize the movement activity for each well at the beginning of the assay (basal, 100% activity). Then, drugs were added to a final volume of 100 μl per well. All drugs were tested with a minimum of 12 replicates per plate. For comparison among different drug concentrations or worm strains, the assays were performed in parallel. Motility values for different experimental groups were analyzed in 5-min time bins during 60 to 120 min. Each condition was evaluated in at least five independent experiments with different synchronized worm batches and always in parallel with the respective control. 5-HT and Tryp solutions were freshly prepared for each assay.
Results
We first explored how 5-HT affects the whole organism by evaluating the response of young adult wildtype worms to5-HT exposure as a function of time and concentration (0.05–4 mM in water) (Fig. 4A). We found that 1 mM 5-HT in water produced a rapid and reversible decrease of motility of wildtype worms. The reduction of motility reached its maximal level, which was between 50 and 80%, after 15 min of exposure to the drug (Fig. 4B, p < 0.001). However, wildtype worms recovered from this quiescent state gradually after 20 min of5-HT exposure in most of the assays. After 60 min, worm motility was close to its basal levels (⁓90% recovery) even in the continued presence of 1 mM 5-HT (Fig. 4A). In contrast, we observed that at 5-HT concentrations higher than 1.5 mM(n = 5), worms did not recover from the paralysis at least during the first 120 min. The mod-1(ok103) mutant strain, which lacks MOD-1, was partially resistant to 1mM5-HT since motility was reduced only about 10 to 20%, thus indicating that MOD-1 is a target for 5-HT motility inhibition (Fig. 4A). The difference of the inhibition between wildtype and mutant worms after 15-min exposure to1 mM 5-HT was statistically significant (p < 0.001, Fig. 4A).The mutant worms also recovered with time, indicating that the reversibility of 5-HT effect was not dependent on MOD-1alone. Furthermore, at higher 5-HT concentrations, (>2 mM) MOD-1 mutant worms were completely paralyzed as wildtype worms. We also explored how Tryp affects young adult C. elegans motility. A clear reduction of worm motility was observed after exposure to 5 mM Tryp. The major effect was achieved after15 min, at which a statistically significant reduction of worm motility occurred (>70%, p < 0.001) (Fig. 4B). The decrease of activity in the continued presence of 5 mM Tryp was more pronounced for wildtype than for mod-1(ok103) worms (⁓72% and⁓50%, respectively; p = 0.006). Thus, as shown for5-HT, MOD-1 is partially involved in the Tryp-paralyzing effect. Differently to 5-HT, worms did not recover to basal activity values during 120 min-exposure (Fig. 4, A and B).
We sought to evaluate if the inhibitory action of PZE on MOD-1 receptor affects the worm response to 5-HT. The first step was to dissect this effect from its agonistic action on muscle UNC-49 (GABAA) receptor that leads to flaccid paralysis. Thus, we tested PZE concentrations at which flaccid paralysis did not occur as revealed by both automatic activity assays and thrashing assays. As shown in Figures 5, no changes in motility were observed at 5 mM PZE after 15 min exposure, whereas at 10 mM PZE, the paralysis was evident. The combination of 1 mM 5-HT, which activates MOD-1, with 5 mM PZE, which inhibits MOD-1, produced smaller motility inhibition than that produced by 1 mM 5-HT alone. These results indicate that PZE attenuates the paralysis induced by exogenous 5-HT (Fig. 5). Thus, these experiments confirm that PZE inhibits MOD-1 in the whole organism.
Journal of Biological Chemistry. RESEARCH ARTICLE| VOLUME 298, ISSUE 9, 102356, SEPTEMBER 2022
Noelia Rodriguez Araujo, Guillermina Hernando, Jeremías Corradi, and Cecilia Bouzat.