An investigation into mode of action and selective toxicity of the novel antiparasitic emodepside

Abstract

Emodepside is a novel anthelmintic with resistance breaking properties. Its mode of action has been extensively studied in the model organism Caenorhadbitis elegans. In this worm emodepside elicits an inhibition of feeding, egg-laying and locomotion, which indicate a potent disruption of normal neuromuscular transmission. Mutagenesis screening for C. elegans resistant to emodepside revealed a calcium activated potassium channel SLO-1 as a target of emodepside. SLO-1 belongs to a family of channels that are highly conserved throughout the animal phyla to regulate cell excitability. This project addressed the question of whether or not the basis for the selective toxicity of emodepside resides in its pharmacological specificity for the nematode SLO-1 dependent signalling pathway. A bioinformatics analysis was used to identify the human channel KCNMA1 as one of the closest mammalian homologues to SLO-1. Two expression systems that enabled the pharmacological characterisation, and comparison, of SLO-1 and KCNMA1 have been established. In the first system plasmids carrying C. elegans slo-1 and human KCNMA1 behind the native pslo-1 promoter, a pan-neuronal promoter, a body-wall muscle promoter or a pharyngeal muscle promoter have been successfully employed for generating transgenic C. elegans expressing either SLO-1 or KCNMA1. These transgenics were deployed to permit functional and pharmacological characterisation of the channels in vivo. Behavioural and electrophysiological assays confirmed that KCNMA1 was functionally expressed in this model organism; observations reinforced by the sensitivity of these strains to mammalian BK channel activators NS1619 and rottlerin. Importantly, with regard to selective toxicity, strains expressing SLO-1 were 100 fold more sensitive to emodepside than strains expressing functional KCNMA1. To more directly assess if emodepside interacts with these channels, SLO-1 and KCNMA1 were expressed in HEK293 cells. Whole cell currents from cells expressing SLO-1 or KCNMA1 were inhibited by a selective inhibitor of mammalian BK channels penitrem A. Activation of SLO-1 currents required micromolar Ca2+ in agreement with previously published data. The experimental conditions required to obtain SLO-1 currents constrained the time-course over which the analysis of emodepside effects could be observed. Nonetheless, these experiments demonstrate emodepside potentiation of both SLO-1 and KCNMA1 currents. Taken together with the genetic evidence this thesis supports the hypothesis that emodepside inhibits neuromuscular activity in C. elegans by facilitating activation of SLO-1. Overall the data support the notion that chemical biology targeted towards SLO-1 channel pharmacophores may provide an important route to resistant breaking anthelmintic drugs.

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Identifiers

ORCID for Anna Crisford: orcid.org/0000-0001-5775-643X

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

ORCID for Vincent O'Connor: orcid.org/0000-0003-3185-5709

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