Kashyap SS et al. (2019), Emodepside has sex-dependent immobilizing effec...

XB-ART-56305

PLoS Pathog 2019 Sep 01;159:e1008041. doi: 10.1371/journal.ppat.1008041.

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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.

Kashyap SS , Verma S , Voronin D , Lustigman S , Kulke D , Robertson AP , Martin RJ .

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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.

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Species referenced: Xenopus laevis
Genes referenced: kcnma1 mef2a

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	Fig 1. Emodepside causes inhibition of motility of whole adult Brugia malayi that is concentration-, time-, and sex-dependent. A: Representative micrograph of an untreated adult female B. malayi. The worm is characteristically coiled and was showing continuous vigorous vibrating movements. B: (female symbol) Concentration- and time-dependent emodepside inhibition of motility on adult B. malayi females and C: (male symbol) males. Motility plotted at 60 minutes for both females and males (D: female symbol: IC50 (female), 801 ± 126nM (SEM)) and males (E: male symbol: IC50 (male), 176 ± 33 nM (SEM)). Each treatment group was normalized to the motility at time 0. N = 12, over two biological replicate experiments. The female IC50 was significantly higher than the male (p<0.001; 2-way ANOVA). The male is more sensitive to emodepside than the female.
	Fig 2. Emodepside produces concentration-dependent outward currents in female and male B. malayi muscle cells. A: Representative current traces of the concentration-dependent emodepside activated standing outward currents from muscle cells of female B. malayi. Cells were whole-cell patch-clamped at -40mV and each concentration of drug was applied consecutively in increasing concentrations (30nM to 3μM) and were allowed to plateau before washing and the next application. B: Emodepside activated outward currents from muscle cells of male B. malayi. C: Concentration-dependent mean current curves for emodepside activated outward currents. Red female symbol: EC50 (Female) = 720 ± 12nM (n = 5), blue male symbol: EC50 (Male) = 294 ± 11nM (n = 5). The male is more sensitive to emodepside than the female.
	Fig 3. slo-1 RNAi treated female worms are resistant to emodepside. A: Knock down of slo-1 transcript after 72 hours of incubation was assayed using qPCR. Knock down of the slo-1 transcript was 91 ± 2% and 22 ± 12% in worms treated with control LacZ dsRNA. (p<0.005, t-test, n = 6). B: Little or no change in Mean Motility Units (MMU) was observed in adult female, Red female symbol, B. malayi control worms, lacZ- and slo-1-dsRNA treated worms over 72 hours. C: Worms soaked in slo-1 dsRNA show an emodepside resistance phenotype. The motility of the control worms was significantly inhibited (only 5% motility was observed after treatment with 300nM emodepside) after 120 minutes while the dsRNA treated worms were resistant to emodepside (2-way ANOVA: p<0.001; n = 24 over 4 biological replicates).
	Fig 4. Emodepside induced outward currents in Xenopus laevis oocyte expressed slo-1 channels. Representative traces for two-electrode voltage clamp (TEVC) experiments on X. laevis oocytes injected with different isoforms of slo-1 cRNA and clamped at +20mV. A: Lack of effects of emodepside on naïve un-injected oocytes. B: Effects of emodepside on B. malayi slo-1f cRNA (15ng) injected oocytes. C: Effects of emodepside on slo-1a cRNA (15ng) injected oocytes. D: Effects of emodepside on slo-1a + slo-1f cRNA (7.5ng each) injected oocytes. E: Concentration-dependent curves for emodepside induced currents from X. laevis oocytes injected with slo-1f (EC50 = 5 ± 1 μM; n = 7), slo-1a and slo-1a+f cRNA (EC50>30μM; n = 7 for both).
	Fig 5. RNAi of slo-1a increases the potency of emodepside. A: Effects of RNAi knock down of slo-1a in adult male. Off target lacZ dsRNA was used as control. IC50s: lacZ Blue male symbol: 181±43nM; slo-1a Blue male symbol = 182±24nM. B: Effects of slo-1a knock-down on adult female. IC50s: lacZ Red female symbol: 617±207nM; slo-1a Red female symbol: 242±75nM. IC50s for both slo-1a (Red female symbol) and (Orange male symbol) were significantly lower than the IC50 of the control lacZ Red female symbol. (2-way ANOVA; p<0.05; N = 8 over two biological replicates). Thus, reduced expression of slo-1a splice variants in females increases the potency of emodepside.


	S1. Effect of voltage-steps on emodepside activated currents in B. malayi muscle cells. A: A representative trace of voltage-activated outward-currents in naïve (black: top) and emodepside treated muscle preparations (red: middle), bottom trace shows the voltage-step protocol (10 mV steps: green), holding potential -40mV. The preparation was perfused with 1μM emodepside for 30 seconds prior to and during the voltage steps. B: Demonstrates the IV plot of control vs emodepside the treated preparation shown in A. C: Shows the activation curve for emodepside mediated mean ±SEM increase in conductance of the potassium channel currents for 5 experiments on 5 preparations like those shown in A and B. Gmax (Emodepside) = 23 ± 1pS, Gmax (Control) = 14 ± 2pS, Vhalf (Emodepside) = -2 ± 1 mV, Vhalf (Control) = 8 ± 1 mV, n = 5. There was little change in the slope factor. Note that Gmax was increased by emodepside showing that the number of SLO-1 channels opening has increased and/or the maximum probability of them being open has increased; the voltage-sensitivity of the channel showed only a modest hyperpolarizing shift.
	S2. Emodepside currents blocked by SLO-1 K channel antagonist, iberiotoxin. A: Representative trace showing the inhibition (reversible on washing) of the emodepside induced current by 100nM iberiotoxin (IbTx). IbTx had no effect on its own. B: Bar chart showing mean ±SEM outward currents in presence of 300 nM emodepside and 300 nM emodepside with 100nM IbTx. IbTX significantly inhibits the outward currents induced by emodepside (p<0.005, paired Student’s t-test, n = 7).

	S4. Selective knock-down of slo-1a transcript in female adult B. malayi. A: Transcript knock-down in female worms specific to slo-1a. Significant knock-down (86.72%) of slo-1a transcript was achieved in female worms while non-specific (lacZ) knock-down of slo-1f was 12.06% (p<0.01, Student’s t-test). n = 5 for each estimation using two biological replicate experiments. B: Shows no slo-1f transcript knock-down in adult male treated with slo-1a specific dsRNA. Male worms lack slo-1a and non-specific knock-down of slo-1f is similar to lacZ dsRNA treated control worms.
	S5. Expression of slo-1 splice variants in female O. volvulus. A: A diagram of the predicted isoforms (splice variants) of slo-1 in O. volvulus and the locations of the primers that were used to amplify the expressed isoforms. B: Table showing the different product sizes for the predicted isoforms when amplified using different the primer combinations. C: Agarose gel showing the expression of slo-1 isoforms in cDNA synthesized from whole worm lysates in female O. volvulus. Amplicons were obtained at 537, 492 and 432bp indicating the expression of slo-1a and slo-1d splice variants.
	S6. Emodepside binding loops on the cytoplasmic domains of SLO-1. Cartoon showing in silico homology modelling of emodepside bound to the RCK regions of other filarial nematodes: A: O. volvulus (Ovo SLO-1A). B: L. sigmodontis (Lsi SLO-1) and C: D. immitis (Dim SLO-1).
	S7. Diagram of a single subunit of SLO-1 illustrating the location of the binding pocket location of emodepside. The SLO-1K channel is composed of a tetramer of subunits each of which have seven transmembrane regions (S0-S6), a pore forming region (P) and a cytoplasmic domain composed of an RCK1 region and an RCK2 region. Only one subunit is displayed. Both the RCK1 and RCK2 regions have calcium-binding sites (★ and ★); RCK1 also has a magnesium-binding region near the emodepside binding site.

References [+] :

Buxton, On the mode of action of emodepside: slow effects on membrane potential and voltage-activated currents in Ascaris suum. 2011, Pubmed