An investigation into the mode of action of the anthelmintic emodepside: a molecular, pharmacological and electrophysiological characterisation

Abstract

Resistance to anthelmintics is increasingly becoming a problem for the agricultural industry. Emodepside is a novel anthelmintic that is effective against a variety of parasitic nematodes in which resistance to other anthelmintics has developed (Harder and Samson-Himmelstjerna, 2002). This suggests that emodepside has a novel mode of action. The aim of this study was to further investigate the mechanism of action of emodepside using the model organism Caenorhabditis elegans. The latrophilin receptors have previously been implicated in the mechanism of action of emodepside. The natural compound from which emodepside is derived (PF1022A) has been shown to bind the Haemonchus contortus receptor latrophilin receptor HC110R (Saeger et al 2001). Caenorhabditis elegans have latrophilin homologues, lat-1 and lat-2. Targeting of lat-1 by RNA interference reduces the sensitivity of the C. elegans pharynx to emodepside (Willson et al 2004). Targeting of either lat-1 or lat-2 by RNA interference reduced the sensitivity of C. elegans to emodepside’s effects on locomotion. Simultaneous targeting of lat-1 and lat-2 by RNA interference reduced the sensitivity of C. elegans to emodepside’s effects on locomotion to a similar extent (Amliwala 2005). RNA interference of latrophilin receptors does not abolish emodepside’s effect, indicating either an incomplete knock down by RNA interference or the presence of an additional mechanism of action. To further investigate role of the latrophilin receptors, the sensitivity of C. elegans mutants carrying deletions in lat- 1(ok1465) and lat-2(tm463) were tested. Neither of these putative null mutants displayed any difference in sensitivity to emodepside’s effects on locomotion compared with the N2 wild type strain. In order to investigate the possibility of a redundancy between lat-1 and lat-2 a lat-2(tm463) lat-1(ok1465) double mutant was constructed. The lat-2(tm463) lat-1(ok1465) double mutant did not display any difference in sensitivity to emodepside’s effects on locomotion compared to N2. However, both lat-1(ok1465) and lat-2(tm463) lat- 1(ok1465) have reduced sensitivity to emodepside’s effects on the pharynx (Bull 2007). This suggests that lat-1 has a role in mediating emodepside’s effects in the pharynx but not in the motor nervous system. Therefore, an alternative mechanism by which emodepside acts in the motor nervous system must exist. The voltage gated calcium sensitive potassium channel slo-1 was previously identified through a mutagenesis screen as a mediator of emodepside’s effect in C. elegans (Amliwala 2005). In this study, further emodepside resistant mutants were isolated. Through the use of genetic complementation tests it was demonstrated that all emodepside resistant mutants are alleles of slo-1. The slo-1(js379) loss of function mutants is highly resistant (insensitive to 10 ?M emodepside) to the effects of emodepside, suggesting emodepside activates SLO-1. Sequencing of slo-1 in mutants isolated for the mutagenesis screens revealed the presence of mutations in regions of SLO-1 involved in the regulation of potassium conductance, supporting the idea that emodepside affects SLO-1 channel activity. slo-1 loss of function mutants are reported to have an increased reversal frequency and an increased sensitivity to the acetylcholine esterase inhibitor aldicarb (Wang et al 2001). Using these phenotypes as a read out of SLO-1 function, the relationship between emodepside sensitivity and SLO-1 function was characterised. In order to confirm a role for slo-1 in the action of emodepside, slo-1(+) was expressed in a slo-1(js379) mutant background. This resulted in restoration of emodepside sensitivity, confirming that SLO-1 mediates emodepside’s effects on locomotion. Expression in either body wall muscle or neurons restored emodepside sensitivity. The effect of emodepside on the rate of aldicarb induced paralysis was investigated. In general, aldicarb hypersensitive worms have increased ACh release and aldicarb resistant worms have reduced ACh release (Mahoney et al 2006). Emodepside reduced the sensitivity of N2 worms to aldicarb, indicating that it causes a reduction in ACh release. These results indicate that emodepside acts on SLO-1 in both muscle and neurons to inhibit locomotion. A direct activation of SLO-1 by emodepside was hypothesised. This was pursued using HEK293 exogenous expression system and patch clamp analysis. The identification of SLO-1 as a target for emodepside provides potential for the development of further anthelmintics as well as the possibility of identifying novel applications for emodepside.

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Identifiers

ORCID for Lindy Holden-Dye: orcid.org/0000-0002-9704-1217

ORCID for Robert Walker: orcid.org/0000-0002-9031-7671

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