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Title: 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}
Article:
Abstract:
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.
Introduction:
Filariae are parasitic nematodes that reside in the definitive host which are always mammals. The adult filariae are referred to as macrofilariae. The adult females release microscopic juveniles, microfilariae, which are then ingested during a blood meal by the arthropod vectors. These microfilariae develop in the arthropod host to the infectious third-stage larvae which can then be transmitted to the mammalian host during a subsequent blood meal. The filaroide species that are responsible for most of the morbidity are Wuchereria bancrofti, Brugia malayi and Brugia timori that cause lymphatic filariasis( including elephantiasis), and Onchocerca volvulus that causes river blindness( onchocerciasis)[1]. These diseases occur mostly in sub-Saharan Africa and Southeast Asia and affect 168 million people. Some 120 million people are infected with lymphatic filariases and around 20 million people are affected by onchocerciasis. The diseases are not usually fatal, but they produce distressing symptoms including, swollen limbs, blindness, reduced work productivity, social rejection and suppression of immune responses to diseases like, malaria and tuberculosis. For over 25 years now, the strategy for controlling filarial diseases has relied on Mass Drug Administration( MDA) programs directed by the World Health Organization using donated drugs to reduce the transmission and morbidity of these filarial diseases. The MDA programs have been successful in several countries, but the filarial diseases still persist, and have not been eliminated[2]. The existing drugs that are now available for MDA( albendazole and ivermectin or diethylcarbamazine for lymphatic filariasis, and ivermectin alone for onchocerciasis are effective against the early larval stages of the parasites( microfilariae), but they do not kill the adults( macrofilariae) efficiently. These repeated annual treatments in MDA programs were modelled to take more than 6–8 years to control lymphatic filariasis, and >15 years to eliminate onchocerciasis because the adults persist and continue to release microfilaria between drug treatments and to spread infection. To accelerate the control measures, there is a real need to identify effective macrofilaricides. The Drugs for Neglected Diseases initiative( DNDi) development program has focused on macrofilaricide drug candidates from repurposing libraries and have included drugs that have been registered for animal health[3]. Emodepside is a veterinary drug that has effects on adult filarial nematodes[4] and has completed first-in-human safety and tolerability studies in healthy volunteers with the purpose of being developed for the treatment of onchocerciasis[5]. Emodepside is a semisynthetic derivative of the cyclooctadepsipeptide PF1022A that was isolated from Rosellinia spp., a fungus that grows on Camellia japonica[6]. Emodepside is effective for treating animal nematode parasites that are resistant to other common anthelmintics[7]. It inhibits motility in nematodes, via an effect that may be mediated through GABAA receptors[8, 9], lathrophilin receptors[10, 11] or SLO-1 K channels[12–15]. The general consensus is that SLO-1 is the main target of emodepside in nematodes[16]. Detailed modes of action studies in filarial nematodes are required to advance the evaluation of emodepside as a macrofilaricide. The effects of emodepside against filariae are dose- and species-dependent. In studies using Litomosoides carinii( syn. L. sigmodontis), Acanthocheinlonema viteae, Brugia malayi, Brugia pahangi, Onchocerca gutturosa and Onchocerca lienalis, the adults of Brugia spp. emerged as the dose-limiting stage where emodepside was least potent[17, 18]. Here we study the mode of action of, and sensitivity to, emodepside using the dose-limiting filarial nematode, B. malayi. RNAi knock down experiments demonstrated that emodepside targets the SLO-1 K channels. We expressed slo-1 isoforms with alternatively spliced exons in the RCK1 domain heterologously in Xenopus laevis oocytes and observed that the slo-1f splice variant, found in muscles of males, is more sensitive to emodepside than the slo-1a splice variant found in muscle of females. We also demonstrate selectively knocking down the slo-1a splice variant in female B. malayi increases emodepside efficacy, signifying a dominant suppressor role for SLO-1A. Further, we show that similar alternatively spliced isoforms are expressed in O. volvulus. In silico modeling identified a putative emodepside binding pocket in the same RCK1 region of different species of filariae that is affected by these splice variations. Our observations show the potent macrofilaricidal effects of emodepside are influenced by sex-linked splice variants in the RCK1 binding pocket of SLO-1. The sex of the filarial target is therefore an important therapeutic variable to consider when evaluating a dose-regimen for treatments during clinical trials[5].
Discussion:
DNDi in collaboration with Bayer AG have pursued the development of emodepside, originally a veterinary anthelmintic, with the aim of it being the first registered oral macrofilaricide treatment for a filarial disease, onchocerciasis, with DNDi planning to run a Phase II “proof-of-concept” clinical trial in sub-Saharan Africa investigating the safety and efficacy of the drug. It has been shown to be effective, albeit with varying potency, against other filarial nematode species in in vitro and in vivo models using rodents[4, 17, 18]. Here we show that emodepside has concentration-dependent activity even against the dose-limiting filarial species, Brugia malayi. An oral dose of 1. 9mg/Kg emodepside produces a maximum plasma concentration of 150μg/L or 125nM in dogs with emodepside being distributed to all organs and the highest concentrations being found in the fat[24]. The IC50 for the adult cattle filaria, O. gutturosa in vitro, is estimated to be <1nM[4]. If 125nM concentrations of emodepside are achieved in humans following treatment, and O. volvulus is as sensitive to emodepside as O. gutturosa, emodepside is anticipated to be effective against onchocerciasis. The effectiveness and safety profile of emodepside against B. malayi has yet to be determined in humans. Our results predict that higher plasma concentrations of emodepside would be required against B. malayi. We find that emodepside was more potent on Bma-SLO-1F than Bma-SLO-1A. We observe that there is alternative splicing in the RCK1 region in B. malayi( slo-1a and slo-1f) and that there are differences in the RCK1 amino acid sequences between the other filarial species as well. In the full-length SLO-1 splice variants of B. malayi and O. volvulus, we find the same alternatively spliced regions between exons 13–20 which encode the RCK1 region. We do not comment more specifically on alternatively spliced slo-1 transcripts of L. sigmodontis due to the lack of sequence information in the database. Although there is a report of splice variants being expressed in D. immitis with alternatively spliced exons in the RCK1 region, very similar to B. malayi[22], we do not have the full-length sequences from the Wormbase Parasite database for D. immitis to use in our study. Nonetheless, we still find less homology between different filarial species in the RCK1 region compared to RCK2( Fig 6). Given these differences in the RCK 1 regions, the reduced potency of emodepside in B. malayi and sensitivity of Onchocerca species, we looked in-silico at the RCK1 region for emodepside binding sites( Fig 6). We found that in all species of filaria examined that emodepside binds most favorably in the same pocket bound by five separate loops, A-E. Loops A-D are present in the RCK1 region( S6 Fig). Loop E is present in the RCK2 region adjacent to the Calcium Bowl. The in silico observations showing emodepside binding to the RCK1 region of O. volvulus further advances our knowledge of its effectiveness as a macrofilaricide for the treatment of river blindness. Observations on a number of C. elegans SLO-1 mutants have also noted that amino-acid changes in the RCK1 region reduces emodepside potency, supporting the presence of an emodepside binding pocket in this region[15, 25]. Our observations do not rule out additional binding of emodepside to transmembrane regions of the channel in the lipid phase of the membrane; but we observed only a small change in the voltage-sensitivity of the channel that would, if large, indicate an effect mediated via the voltage-sensing domain in the membrane[26, 27]. We have observed that emodepside is more potent on male B. malayi than females and therefore the gender of nematodes has an effect as a variable on the outcome of emodepside treatment. Insufficient concentrations of emodepside would leave the female worms surviving treatment to continue their life cycle and the diseases. The National Institutes of Health( NIH) expects that scientists test for the possible role of gender as a biological variable in vertebrate animals and humans( Notice Number NOT-OD-15-102) but does not require reporting of gender as a variable on invertebrates, here parasitic nematodes. This study shows that gender is a significant factor for the effect of emodepside. Significant gender-dependent differences in the metabolism of anthelmintics has been observed for Haemonchus contortus[28] with females metabolizing benzimidazole anthelmintics more extensively than males and resistance of H. contortus females showing greater sensitivity to ivermectin than males[29] but gender-linked effects are not found routinely[30]. We advocate that gender, as a variable for parasitic nematodes should be investigated as early as possible because of potential therapeutic consequences.
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 .