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, the introduction of drugs with adulticidal activity is paramount. The broad-spectrum anthelmintic emodepside, a nematode-selective SLO-1 K channel activator, has been considered 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. Ovo-SLO-1A was more sensitive to emodepside than Bma-SLO-1F. GoSlo-SR-5-69 was a positive allosteric modulator, potentiating the effects of emodepside. Emodepside was docked at the S6 pocket below the selectivity filter for Bma-SLO-1F and Ovo-SLO-1A. The binding of emodepside in the S6 pocket indicated a stabilizing π-π interaction between F342 and the phenyl rings of emodepside which may contribute to the potency of emodepside on these filaria channels. Molecular docking suggested that 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.

Sequence Analysis

Bma-SLO-1F and Ovo-SLO-1A amino acid sequences were acquired from the B. malayi and O. volvulus genomes using WormBase ParaSite (parasite.wormbase.org). Sequence alignment was conducted using EMBOSS Needle pairwise sequence alignment tools, with EBLOSUM62 matrix, a default gap penalty of 10, and extension penalty of 0.5, to determine sequence identity and similarity between both species and isoforms.

Sequence annotation was achieved using previously published alignment information by highlighting the voltage sensor domain (VSD), pore domain (PD), and the regulator of potassium conductance domains (RCK1 and RCK2). To further estimate conservation of each individual domain between isoforms, alignments, and sequence identity analysis were also conducted for each domain individually.

Cloning of Brugia malayi slo-1f and Onchocerca volvulus slo-1a

Primers for Brugia malayi slo-1f and Onchocerca volvulus slo-1a isoforms were designed with sequences flanking the pT7TS-rich expression vector that included the restriction site (NheI). PCR amplification was conducted on B. malayi slo-1f that was previously cloned in the pCDNA3.1 vector. In contrast, Onchocerca volvulus slo-1a was synthesized by Life Technologies GeneArt. Subsequently, both amplicons were separated on a 1% Agarose SYBR Safe gel, purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel) and cloned into the pT7TS-rich vector by using Infusion HD Cloning Kit (Takara Bio USA, Inc) according to the manufacturer’s protocols. Once cloned, the plasmids were verified by sequencing.

The pT7TS-rich plasmids containing cloned products of either Bma-slo-1f or Ovo-Slo-1a were linearized by SmaI and BamHI respectively and purified. Capped cRNAs were then synthesized from the linearized vectors containing the B. malayi and O. volvulus Slo-1 isoforms previously mentioned using the T7 mMessage mMachine Kit (Ambion, USA). The cRNAs were stored at -80°C until further use.

Heterologous expression of Bma-SLO-1F and Ovo-SLO-1F receptors in Xenopus laevis oocytes

Defolliculated Xenopus laevis oocytes were purchased from Ecocyte Bioscience (Austin, TX, USA) and Xenopus 1 Corp (Dexter, MI, USA). Heterologous expression of the Bma-SLO-1F receptor was achieved by injecting 15 ng of cRNA in a total volume of 50 nL in nuclease-free water. Each oocyte was microinjected into the cytoplasm of the animal pole region using a Drummond Nanoject II microinjector (Drummond Scientific, Broomall, PA, USA). After injection, oocytes were incubated at 17°C in a sterile 96-well culture plate containing 300 μl of incubation solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂.2H₂O, 1 mM MgCl₂.6H₂O, 5 mM HEPES, 2.5 mM Na pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin, pH 7.5) in each well. Incubation solution was changed daily during the period of incubation. The same procedure was also conducted for the Ovo-SLO-1A receptor. Experiments were performed on oocytes within 5 – 6 days post injection.

Two-microelectrode voltage clamp (TEVC) electrophysiology

TEVC was conducted at room temperature by impaling oocytes with two microelectrodes; a current injecting electrode, Im, used to inject the required current for holding the membrane at a set voltage, and a voltage sensing electrode, Vm. The microelectrodes were pulled using a Flaming/Brown horizontal electrode puller (Model P-97; Sutter Instruments, Novato, CA, USA) and filled with 3 M KCl. Each electrode tip was broken with a piece of Kimwipe paper (Kimtech Science^TM, Fisher) to achieve a resistance of 2 – 5 MΏ in recording solution (88 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂.6H₂O, 1.8 mM CaCl₂.2H₂O and 5 mM HEPES, at pH 7.4). To investigate the concentration-response relationship of emodepside on the expressed Bma-SLO-1F or Ovo-SLO-1A receptors, oocytes were voltage clamped at a steady-state potential of +20 mV with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA). Amplified signals were converted from analog to digital format by a Digidata 1322A digitizer (Molecular Devices, CA, USA) and all data were acquired on a desktop computer with the Clampex 10.3 data acquisition software (Molecular Devices, Sunnyvale, CA, USA). In addition, the same protocol was also used to test the effects of GoSlo-SR-5-69 alone or in combination with emodepside.

Voltage Step Electrophysiology

Voltage step experiments were conducted using the two-electrode voltage-clamp technique to determine current-voltage relationships of each receptor in the absence and presence of 1 µM emodepside. Briefly, oocytes expressing either Bma-SLO-1F or Ovo-SLO-1A channels were impaled and subjected to a current-voltage protocol that consisted of 500 ms voltage steps from -120 to + 60 mV in 20 mV increments, starting from a holding position of -70 mV for 1 s between each step. Plateau currents were recorded at a frequency of 5000 Hz during clamping and perfused with recording solution: (88 mM NaCl, 2.5 mM, KCl, 1 mM MgCl₂.6H₂O, 1.8 mM CaCl₂.2H₂O and 5 mM HEPES, at pH 7.4). The results of the voltage steps were evaluated and analyzed using the ClampFit 10.3 software (Molecular Devices), whereas current-voltage curves (IVCs) were prepared using Graphpad Prism 10.1.1(GraphPad Software, Inc., USA).

Chemicals

Emodepside was purchased from Advanced ChemBlock Inc (Hayward, CA, USA). GoSlo-SR-5-69 was purchased from Tocris Bioscience (Bristol, UK). Stock solutions of emodepside were prepared at 0.1, 0.3, 1, 3, 10 and 30 mM in dimethyl sulfoxide (DMSO) solutions prior to experimentation, then diluted in recording solution. Stock solutions of GoSlo-SR-5-69 were made in DMSO, at 50 mM, then diluted in recording solution to the required concentrations (3 µM). The final DMSO concentration did not exceed 0.1% in the experimental solutions.

Drug application

Emodepside is known to be lipophilic, thus making it difficult to wash off completely from the Xenopus laevis oocyte preparation after application. Additionally, emodepside concentrations exceeding 10 µM showed evidence of precipitation and a limit of solubility. To estimate EC₅₀ values, we utilized a cumulative concentration-response protocol (no wash steps between drug application) and a maximum concentration of 10 µM emodepside. For our recordings, the times for drug applications were selected to allow the currents recorded to reach a stable plateau. At the beginning of experiments the Xenopus oocytes were perfused with drug free recording solution for 1 min, to obtain stable initial resting currents. For our drug applications we used successive applications of increasing concentrations of emodepside (0.1 – 10 µM) for 5 mins per concentration. Washing of the Xenopus oocytes then followed for 3 mins.

The effects of the mammalian BK channel blocker, verruculogen was tested on Bma-SLO-1F and Ovo-SLO-1A expressing oocytes in a manner before and after the application of emodepside. Briefly, oocytes were perfused for 1 min with recording solution which was then followed by 2 mins application of verruculogen and subsequently application of emodepside for 5 mins. These experiments were repeated in the opposite order where emodepside was applied first for 5 mins, followed by verruculogen for 2 mins.

To investigate the effects of GoSlo-SR-5-69 (a mammalian BK channel activator), on Bma-SLO-1F and Ovo-SLO-1A channels, oocytes were perfused with recording solution for 1 min followed by application of 3 µM GoSlo-SR-5-69 for 5 mins and subsequently 0.3 µM emodepside (positive control) for 5 mins with a final wash step of recording solution for 3 mins.

To determine the effects of GoSlo-SR-5-69 in combination with emodepside, we employed a protocol for oocytes that involved 1 min perfusion of recording solution to obtain the control current levels. This was followed by the application of 0.3 µM emodepside for 5 mins, then co-application of 3 µM GoSlo-SR-5-69 in the continued presence of 0.3 µM emodepside for 5 mins and immediate wash off with 0.3 µM emodepside for 5 mins and a final wash with recording solution for 3 mins.

Finally, to evaluate the effects of 3µM GoSlo-SR-5-69 on emodepside concentration-response relationships, recording solution was applied for 1 min to each oocyte, followed by 3 µM GoSlo-SR-5-69 until evoked currents were stabilized. Next, 5 mins applications of increasing concentrations of emodepside (0.1 – 10 µM) were perfused in the continued presence of 3 µM GoSlo-SR-5-69. A 3 min wash off time was allowed at the end of the final concentration of emodepside.

Homology Modelling and Molecular Docking

SLO-1 channels for O. volvulus and B. malayi were constructed from FASTA sequences obtained from the WormBase genome projects (Bma-SLO-1F and Ovo-SLO-1A). Homology models were constructed using SWISS-MODEL, with the Drosophila melanogaster SLO channels in Ca²⁺ bound state (RCSB: 7PXE) as template. The structure of emodepside was obtained from PubChem (CID6918632), and pre-docking structures of the channels were energy-minimized using GROMACS 2023.2, solvated in water and neutralized with potassium (K^+^) ions. AMBER99SB-ILDN force field was used, and water molecules were parametrized with SPC/E. Emodepside was parameterized with GAFF using Antechamber version 17.3. The structure of GoSlo-SR-5-69 was obtained from PubChem (CID56944133), and prepared for docking using LigPrep in the Schrödinger package version 2023-4 at pH 7.0, with OPLS4 FF.

Docking was performed using GLIDE module in Schrödinger, docked to the homology models of SLO-1A for O. volvulus, SLO-1F for B. malayi and the cryo-EM structure for D. melanogaster SLO. Docking for all structures was performed at Extended Precision (XP) level. The S6 emodepside pocket was defined as the position emodepside adopted in the energy-minimized structure. The RCK1 binding site was defined as the centroid of G430, M433, Y377, D411 for O. volvulus and B. malayi and for the RCK2, L777, H491, Y490, R781 for B. malayi and L792, H491, Y490, and R796 for O. volvulus.

Analysis of docking results was performed in the Schrödinger Maestro suite to identify non-covalent interactions between ligand and receptor, RMSD was calculated by aligning the structures in PyMOL (v.2.5.4).

Data analysis

Our emodepside concentration-response experiments involved the use of Clampfit 10.3 (Molecular Devices, Sunnyvale, CA, USA) to measure peak current responses for each drug concentration (0.1 - 10 µM) per oocyte. GraphPad Prism 10.1.1 software (GraphPad Software Inc., USA) was used to generate Concentration-response curves using the log agonist vs. response equation (variable slope) to estimate EC₅₀, R_max, and Hillslope (nH) values for both Bma-SLO-1F and Ovo-SLO-1A channels. We also used the unpaired two-tailed Student’s T-test to test for statistical significance. A p value < 0.05 was deemed significant. The analyzed results were expressed as mean ± S.E.M.

To obtain current-voltage curves (IVCs) from our voltage steps experiments, plateau currents elicited at each step potential (-120 to + 60 mV) were acquired for individual oocytes in Clampfit 10.3 (Molecular Devices, Sunnyvale, CA, USA). Mean currents for all replicate oocytes were plotted against their corresponding voltage step potentials to obtain IVCs using Graphpad Prism 10.1.1 software (GraphPad Software, Inc., USA). Mean currents between each treatment group of oocytes were compared for each step potential using two-way ANOVA and Tukey’s multiple comparison test to test for significance. To obtain and compare conductance changes in the absence and presence of 1 µM emodepside for Bma-SLO-1F and Ovo-SLO-1A channels, IVCs were analyzed for slopes between step potentials of -120 and +60 mV using linear regression analysis in Graphpad Prism 10.1.1 (GraphPad Software, Inc., USA). Subsequently, statistical differences for slope values among treatment groups were analyzed using ANOVA, followed by the Tukey multiple comparison post-hoc test. Results were expressed as mean ± S.E.M.

To determine statistical significance of emodepside potentiation by GoSlo-SR-5-69, we measured the mean currents evoked by 0.3 µM emodepside alone and compared it to the subsequent application of 3 µM GoSlo-SR-5-69 in combination with 0.3 µM emodepside on each oocyte using the unpaired two-tailed Student’s t-test in GraphPad Prism 10.1.1. Similar analyses were also conducted for the effect of extracellular Ca²⁺ on GoSlo-SR-5-69 potentiation of emodepside response involving recordings conducted in normal recording solution (1.8 mM added Ca²⁺) and modified recording solution (Ca²⁺-free). The results were expressed as the mean ± S.E.M.

Analysis involving the determination of GoSlo-SR-5-69 effects on Bma-SLO-1F and Ovo-SLO-1A mediated emodepside concentration-response relationship were conducted in a similar manner as previously described for the emodepside concentration-response experiments. Student’s t-tests were also used for comparing EC_50s and R_max for emodepside alone and emodepside in combination with GoSlo-SR-5-69 using GraphPad Prism 10.1.1 software (GraphPad Software, Inc., USA). Results were expressed as the mean ± S.E.M.