An investigation into the mechanism of action of emodepside and other novel anthelmintic compounds

Abstract

Anthelmintic drugs are used as a control measure for parasitic nematode infections. However, heavy reliance on these compounds has led to the development of parasite resistance and a consequent need to develop new anthelmintics that function via novel biological pathways. In this project, seven potential anthelmintics were examined for activity in the free-living nematode C. elegans. Of the seven compounds, those belonging to the cyclooctadepsipeptide group were shown to inhibit C. elegans pharyngeal pumping. The most potent of the compounds tested was emodepside, which has previously been shown to act as a potent broad-spectrum anthelmintic. The major aim of this project was to define and characterise the mechanism of action of emodepside using the free-living nematode C. elegans. Emodepside potently inhibited the locomotion and pharyngeal pumping of adult and larval stage 4 (L4) C. elegans. The L4 pharynx demonstrated reduced emodepside sensitivity in the presence, but not absence, of the intact cuticle, suggesting that the L4 cuticle reduces drug access. Continuous exposure of C. elegans to emodepside from egg to adult resulted in a slowing of worm development, possibly due to the paralysis of locomotion and pharyngeal pumping, which are important for food location and ingestion. Emodepside inhibited C. elegans egg laying behaviour, but not egg production or egg hatching. These results suggest that emodepside functions at the neuromuscular junction of the pharyngeal, vulval and body wall muscles to produce paralysis of pharyngeal pumping, egg laying and locomotion. Analysis of specific C. elegans gene mutants for their sensitivity to the inhibition of pharyngeal 5-HT response by emodepside strongly suggested that the anthelmintic functions primarily via the Ca2+- activated K+ channel SLO-1, with minor contributory roles performed by the G protein-coupled receptor LAT-1, and the G proteins Go;, and G(V The C. elegans slo-1 (js379) null demonstrated a high level of resistance to emodepside, which was abolished upon rescue of slo-1 expression in neurons. Rescue of slo- 1 expression in the pharyngeal muscles did not restore emodepside sensitivity, suggesting that neuronal SLO-1 is activated by emodepside to achieve paralysis of pharyngeal pumping. A gain-of-function mutant for SLO-1 that increases the Ca2+ sensitivity of the channel was found to be hypersensitive to emodepside, suggesting that emodepside manipulates the Ca2+ sensitivity of SLO-1 to facilitate its activity. To establish how emodepside is affecting SLO-1, the role of this channel in the pharynx was investigated. The s/o-7 Qs379) null possessed a longer mean pump duration and a disrupted pattern of pumping. Rescue of neuronal SLO-1 restored the wild type phenotype, suggesting that neuronal SLO-1 contributes to the control of pharyngeal pumping via the regulation of neurotransmitter release. Therefore, it is possible that emodepside may function by increasing the Ca2+ sensitivity of neuronal SLO-1 at the pharyngeal neuromuscular junction, resulting in an inhibition of neurotransmitter release and muscle paralysis. However, whole cell patch clamp recordings from pharyngeal muscle cells showed that emodepside inhibits SLO-1 currents rather than activating them. It is possible that the different intracellular environments afforded by pharyngeal neurons and muscle may result in the SLO-1 channel being modified differently in these two locations. This could produce a neuronal channel that is stimulated by emodepside and a muscle channel inhibited by the drug. Interestingly, the lat-1 (okl465) mutant was resistant to the effect of emodepside on pharyngeal pumping but not locomotion, suggesting that components of the molecular pathways involved in emodepside activity at the pharyngeal and body wall muscle neuromuscular junctions may be subtly different. The cyclooctadepsipeptides verticilide, PF1022-222 and PF1022-888 were also shown to inhibit C. elegans pharyngeal pumping. The slo-1 (js379) and lat-1 (okl465) null mutants demonstrated reduced sensitivity to all three compounds, suggesting that SLO-1 and LAT-1 are involved in the mechanism of action of these compounds as well as emodepside. SLO-1 and LAT-1 are not known to be involved in the mechanism of action of any other currently available anthelmintic, highlighting the novel functioning of the compounds tested in this project, and their potential as anthelmintics that could break resistance.

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