McHugh M et al. (2025), Positive allosteric modulation of emodepside se...

XB-ART-61517

PLoS Pathog 2025 Sep 11;219:e1012946. doi: 10.1371/journal.ppat.1012946.

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Positive allosteric modulation of emodepside sensitive Brugia malayi SLO-1F and Onchocerca volvulus SLO-1A potassium channels by GoSlo-SR-5-69.

McHugh M, Njeshi CN, Smith N, Kashyap SS, Datta R, Sun H, Robertson AP, Martin RJ.

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Human lymphatic filariasis and onchocerciasis are Neglected Tropical Diseases (NTDs), of major public health concern. Prophylaxis and treatment rely on anthelmintics that effectively eliminate migrating microfilariae but lack efficacy against adult filarial worms. To expedite the elimination of both diseases, drugs with adulticidal activity are needed. The broad-spectrum anthelmintic emodepside, a nematode selective SLO-1 K channel activator, is a promising candidate for the treatment of onchocerciasis due to its macrofilaricidal activity against Onchocerca volvulus. Nevertheless, it is less effective against adult Brugia malayi, one of the causative agents of human lymphatic filariasis. Characterizing molecular and pharmacological disparities between highly conserved splice variant isoforms of B. malayi and O. volvulus SLO-1 K channels and identifying allosteric modulators that can increase emodepside potency on B. malayi SLO-1 K channels is necessary for therapeutic advance. In this study, we tested the effects of emodepside and the mammalian BK channel activator, GoSlo-SR-5-69 alone and in combination on Xenopus expressed B. malayi SLO-1F and O. volvulus SLO-1A channels. Additionally, binding poses of emodepside, and GoSlo-SR-5-69 were predicted on both channels using molecular docking. We observed that Ovo-SLO-1A was more sensitive to emodepside than Bma-SLO-1F, with EC50 values of 0.40 ± 0.05 µM and 1.4 ± 0.2 µM for Ovo-SLO-1A and Bma-SLO-1F respectively. GoSlo-SR-5-69 lacked agonist activity on both channel isoforms but acted as a positive allosteric modulator, potentiating the effects of emodepside. Molecular docking analysis revealed that emodepside binds at the S6 pocket below the selectivity filter for Bma-SLO-1F and Ovo-SLO-1A. In contrast, GoSlo-SR-5-69 binds at the RCK1 pocket. This study reveals for the first time, allosteric modulation of filarial nematode SLO-1 K channels by a mammalian BK channel activator and highlights its ability to increase emodepside potency on the B. malayi SLO-1 K channel.

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Species referenced: Xenopus laevis
GO keywords: potassium channel activity

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	Fig 1. Amino acid sequence alignment of Bma-SLO-1F and Ovo-SLO-1A.The voltage sensor domain (VSD; orange boxes), pore domain (PD; blue boxes) comprising the selectivity filter (light blue box) and two C-terminal domains for regulator of K+ conductance (RCK1; pink boxes and RCK2; black boxes) are indicated. Amino acids which are not identical between filarial species are highlighted by a black background. Gaps are indicated by “_” symbols for amino acid residues that are missing.
	Fig 2. Emodepside (emo) concentration response relationships for Ovo-SLO-1A and Bma-SLO-1F.A. Representative traces for two-electrode voltage-clamp recording showing outward currents for Ovo-SLO-1A (top; pink trace) and Bma-SLO-1F (lower; blue trace) channels, elicited in the presence of increasing concentrations of emodepside (0.1 to 10 µM) at a holding potential of +20 mV. B. Emodepside concentration-response relationships for Ovo-SLO-1A (pink) and Bma-SLO-1F. C. Emodepside EC50 analysis (mean ± S.E.M) for Ovo-SLO-1A and Bma-SLO-1F channels. D: Maximum current responses (Rmax) (mean ± S.E.M) of emodepside for Ovo-SLO-1A and Bma-SLO-1F channels. Bottom was constrained to zero for curve fitting. Emodepside concentration-response curves were generated using n = 10 oocytes for Ovo-SLO-1 A and n = 10 oocytes for Bma-SLO-1F, pooled from three independent batches of oocytes to generate 10 biological replicates. EC50 and Rmax values were also prepared using the concentration response curve analysis. *P < 0.001, **P < 0.0001 significantly different as indicated; unpaired two-tailed student t-test.
	Fig 3. Effects of emodepside on current-voltage curves (IVCs) of the Ovo-SLO-1A and Bma-SLO-1F channels expressed in Xenopus laevis oocytes.A. Basal currents (mean ± S.E.M) from oocytes expressing Ovo-SLO-1A (n = 6 biological replicates, black) or injected with water (n = 6 biological replicates, grey) in the absence of emodepside. Currents (mean ± S.E.M) obtained from oocytes expressing Ovo-SLO-1A (n = 6 biological replicates, pink) or injected with water (n = 6 biological replicates, red) in the presence of 1 µM emodepside. B. Slope conductance analysis (mean ± S.E.M) of Ovo-SLO-1A expressing oocytes perfused with recording solution and no drug (n = 6 biological replicates; black), Ovo-SLO-1A expressing oocytes exposed to 1 µM emo (n = 6 biological replicates; pink), water injected oocytes perfused with recording solution and no drug (grey; n = 6 biological replicates) and water injected oocytes exposed to 1 µM emodepside (red; n = 6 biological replicates). C. Basal currents (mean ± S.E.M) from oocytes expressing Bma-SLO-1F (n = 6 biological replicates, black) or injected with water (n = 6 biological replicates, grey) in the absence of emodepside. Currents (mean ± S.E.M) obtained from oocytes expressing Bma-SLO-1F (n = 6 biological, blue) or injected with water (n = 6 biological replicates, tan) in the presence of 1 µM emodepside. D. Slope conductance analysis (mean ± S.E.M) of Bma-SLO-1F expressing oocytes perfused with recording solution and no drug (n = 6 biological replicates; black), Bma-SLO-1F expressing oocytes exposed to 1 µM emo (n = 6 biological replicates; pink), water injected oocytes perfused with recording solution and no drug (grey; n = 6 biological replicates) and water injected oocytes exposed to 1 µM emodepside (red; n = 6 biological replicates). Biological replicates were pooled from two independent studies for water injected, Bma-SLO-1F and Ovo-SLO-1A injected oocytes.
	Fig 4. Effects of GoSlo-SR-5-69 on Bma-SLO-1F and Ovo-SLO-1A channels.A. Sample trace of water injected oocytes (n = 6). B. Sample trace of oocytes expressing the Bma-SLO-1F channel (n = 6). C. Sample trace of Ovo-SLO-1A expressing channel (n = 6). Application of 3 µM GoSlo-SR-5-69 produced transient inactivating inward currents in oocytes injected with water, Bma-SLO-1F and Ovo-SLO-1A channels. Emodepside failed to activate water injected oocytes but produced outward currents in oocytes expressing Bma-SLO-1F and Ovo-SLO-1A channels. For each splice variant and water injected oocytes, 6 oocytes were tested from a single batch to generate 6 biological replicates.
	Fig 5. Effects of GoSlo-SR-5-69 on Bma-SLO-1F and Ovo-SLO-1A-mediated emodepside responses.A. Representative traces for Bma-SLO-1F, Ovo-SLO-1A and water injected oocytes perfused with 0.3 µM emodepside, followed by 3 µM GoSlo-SR-5-69 in the continued presence of emodepside and an initial wash with 0.3 µM emodepside with a final wash with oocyte recording solution. B. Mean current responses (in nA) generated in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 for oocytes expressing the Bma-SLO-1F channel. Blue bar: 0.3 µM emodepside alone (n = 6 biological replicates, pooled from two independent experiments). Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from two independent experiments). C. Mean current responses (in nA) generated in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 for oocytes expressing the Ovo-SLO-1A channel. Pink bar: 0.3 µM emodepside alone (n = 6 biological replicates, pooled from two independent experiments). Black bar: 3 µM GoSlo-SR-5-69 in combination with 0.3 µM emodepside (n = 6 biological replicates, pooled from two independent experiments). D. Percentage (%) increase analyses of currents produced by emodepside and GoSlo-SR-5-69 co-application for Bma-SLO-1F (white, n = 6) and Ovo-SLO-1A (grey, n = 6). Data are plotted as mean ± S.E.M; **P < 0.0001 significantly different as indicated; paired two-tailed student t-test; P < 0.001, significantly different as indicated, unpaired two-tailed student t-test.
	Fig 6. Effects of extracellular Ca2+ on GoSlo-SR-5-69 potentiation of Bma-SLO-1F and Ovo-SLO-1A-mediated emodepside responses.A. Peak current (in nA) amplitudes produced by Bma-SLO-1F expressing oocytes in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the presence of normal recording solution consisting of 1.8 mM added Ca2+. Blue bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). **P < 0.0001 significantly different as indicated; paired two-tailed student t-test B. Peak current (in nA) amplitudes produced by oocytes expressing the Bma-SLO-1F channel in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the absence of 1.8 mM added Ca2+. Blue bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). P < 0.001, significantly different as indicated, paired two-tailed student t-test. C. Percentage (%) increase in currents produced by emodepside and GoSlo-SR-5-69 co-application for Bma-SLO-1F expressing oocytes perfused with normal recording solution, containing 1.8 mM added Ca2+ (White bar with brown border, n = 6), or modified recording solution lacking added Ca2+(grey bar with black border, n = 6). P > 0.05, no statistical significance (ns) as indicated, unpaired two-tailed student t-test. D. Peak current (in nA) amplitudes produced by oocytes expressing the Ovo-SLO-1A channel in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the presence of 1.8 mM added Ca2+. Pink bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). E. Peak current (in nA) amplitudes produced by Ovo-SLO-1A expressing oocytes in response to 0.3 µM emodepside alone and in combination with 3 µM GoSlo-SR-5-69 in the absence of 1.8 mM added Ca2+. Pink bar: 0.3 µM emodepside alone; Black bar: 3 µM GoSlo-SR-5-69 co-applied with 0.3 µM emodepside (n = 6 biological replicates, pooled from three independent experiments). P < 0.0001 significantly different as indicated; paired two-tailed student t-test; **P < 0.01, significantly different as indicated, paired two-tailed student t-test. F. Percentage (%) increase in currents produced by emodepside and GoSlo-SR-5-69 co-application for Ovo-SLO-1A expressing oocytes in the presence of 1.8 mM added Ca2+ (White bar with orange border, n = 6), or modified recording solution lacking added Ca2+(Grey bar with black border, n = 6). P > 0.05, no statistical significance (ns) as indicated, unpaired two-tailed student t-test.
	Fig 7. Effects of GoSlo-SR-5-69 as a positive allosteric modulator on Bma-SLO-1F and Ovo-SLO-1A channels on emodepside mediated response.A. Concentration-response plots for emodepside alone, (blue) and emodepside in the presence of 3 µM GoSlo-SR-5-69 (black) for the Bma-SLO-1F channel, (n = 6 biological replicates, pooled from three independent experiments). B. Emodepside concentration-response plots for Ovo-SLO-1A in the presence of emodepside alone, pink) and emodepside in the presence of 3 µM GoSlo-SR-5-69 (black), (n = 6 biological replicates, pooled from three independent experiments). Bottom was constrained to zero for curve fitting.
	Fig 8. Identified poses of emodepside and GoSlo-SR-5-69 in Ovo-SLO-1A and Bma-SLO-1F channels.A. Emodepside bound at the S6 pocket for Ovo-SLO-1A channel. B. GoSlo-SR-5-69 bound at the RCK1 pocket for Ovo-SLO-1A channel. C. Emodepside bound at the S6 pocket for Bma-SLO-1F channel D. GoSlo-SR-5-69 bound at the RCK1 pocket for Bma-SLO-1F channel. For both channels, emodepside is bound below the selectivity filter, indicating the π-π stacking between F342 and emodepside. Displayed are also the stabilizing ligand-receptor interactions between GoSlo-SR-5-69 and the respective filarial nematode splice variant channels.
	Fig 9. Summary diagram of the putative mechanism of GoSlo-SR-5-69 alone and in combination with emodepside on Bma-SLO-1F and Ovo-SLO-1A channels.A. Emodepside (gold ring) binds within the pore domain (PD) beneath the selectivity filter (light blue inverted block arc) and directly activates either Bma-SLO-1F or Ovo-SLO-1A channels resulting in the translocation of K+ ions (red circles) out of the cell (outward current). B. Activation of Bma-SLO-1F or Ovo-SLO-1A by emodepside (gold ring) in the PD. GoSlo-SR-5-69 (green octagon) then binds to a putative allosteric site (RCK1 domain) of the channel giving rise to stabilization of the channel gating ring in the open state. Subsequently, K+ permeation and translocation are further increased leading to greater current amplitude and the potentiation of emodepside response. The removal of GoSlo-SR-5-69 results in ion translocation almost back to normal levels previously seen in the presence of emodepside binding alone.