Emodepside has sex-dependent immobilizing effects on adult Brugia malayi due to a differentially spliced binding pocket in the RCK1 region of the SLO-1 K channel

Filariae are parasitic nematodes that are transmitted to their definitive host as third-stage larvae by arthropod vectors like mosquitoes. Filariae cause diseases including: lymphatic filariasis with distressing and disturbing symptoms like elephantiasis; and river blindness. Filarial diseases affect millions of people in 73 countries throughout the topics and sub-tropics. The drugs available for mass drug administration, (ivermectin, albendazole and diethylcarbamazine), are ineffective against adult filariae (macrofilariae) at the registered dosing regimen; this generates a real and urgent need to identify effective macrofilaricides. Emodepside, a veterinary anthelmintic registered for treatment of nematode infections in cats and dogs, is reported to have macrofilaricidal effects. Here, we explore the mode of action of emodepside using adult Brugia malayi, one of the species that causes lymphatic filariasis. Whole-parasite motility measurement with Worminator and patch-clamp of single muscle cells show that emodepside potently inhibits motility by activating voltage-gated potassium channels and that the male is more sensitive than the female. RNAi knock down suggests that emodepside targets SLO-1 K channels. We expressed slo-1 isoforms, with alternatively spliced exons at the RCK1 (Regulator of Conductance of Potassium) domain, heterologously in Xenopus laevis oocytes. We discovered that the slo-1f isoform, found in muscles of males, is more sensitive to emodepside than the slo-1a isoform found in muscles of females; and selective RNAi of the slo-1a isoform in female worms increased emodepside potency. In Onchocerca volvulus, that causes river blindness, we found two isoforms in adult females with homology to Bma-SLO-1A and Bma-SLO-1F at the RCK1 domain. In silico modeling identified an emodepside binding pocket in the same RCK1 region of different species of filaria that is affected by these splice variations. Our observations show that emodepside has potent macrofilaricidal effects and alternative splicing in the RCK1 binding pocket affects potency. Therefore, the evaluation of potential sex-dependent effects of an anthelmintic compound is of importance to prevent any under-dosing of one or the other gender of nematodes once given to patients.
Author summary Emodepside is an anthelmintic drug that is used for treatment of nematode infections in cats and dogs. A potassium channel, SLO-1, has been implicated as a target for emodepside in the nematode model Caenorhabditis elegans. The anthelmintic effects of emodepside has been demonstrated against filariae, but its molecular mode of action requires further elucidation. Here, we show that emodepside activates SLO-1 channels in the human filaria, Brugia malayi, one of the causative agents of lymphatic filariasis. Emodepside is more potent on male than on the female B. malayi. Worms where slo-1 was knocked down were less susceptible to emodepside. Alternative splicing of the slo-1 gene in the region encoding the RCK1 domain, results in the expression of SLO-1F and SLO-1A in female worms and SLO-1F alone in male worms. SLO-1F expressed in Xenopus laevis oocytes was more sensitive to emodepside than SLO-1A or a combination of SLO-1A and SLO-1F. Selective knockdown of slo-1a in female worms increased their emodepside sensitivity, so their sensitivity resembled male worms. Emodepside binds to the RCK1 region of SLO-1 in in-silico protein interaction studies of the SLO-1 sequences of different filariod species. This is the first report demonstrating a gender-specific effect of emodepside correlating directly to the expression of alternatively spliced isoforms of SLO-1. This study emphasizes that the gender-dependent effects should always be considered when evaluating the efficacy profile of an anthelmintic drug candidate.