An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules

Abstract

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

Keywords: Antibody discovery; Antivenom; Drug discovery; Snake venom neurotoxin; Three-finger toxin; nicotinic acetylcholine receptor (nAChR); α-neurotoxins.

Graphical abstract

Fig. 1

Schematic of the neuromuscular junction and overview of corresponding readouts from the developed assay of nAChR activation. The schematics of the neuromuscular junction (top panels) demonstrate the release of ACh from the pre-synaptic neuron and binding of ACh to the post-synaptic membrane in the absence of α-NTxs (left, Agonist), in the presence of α-NTxs (middle, Antagonist), and in the presence of both α-NTxs and α-NTx-inhibitors (right, α-NTx-inhibitor). Underneath each schematic is the typical fluorescent response using the assay of a well in a 96-well plate containing TE671 cells when ACh is applied with different pre-incubation conditions after a 20 s baseline recording. The three conditions represent the responses when assay buffer alone (Agonist), α-NTx (Antagonist) and α-NTx plus α-NTx-inhibitors (α-NTx-inhibitors: antibodies, decoy receptors, and small molecules) are added. All schematics were created with BioRender.com.

Fig. 2

Known nAChR agonists and antagonists show expected action on TE671 nAChR activation when using membrane potential dye. (A) Representative traces showing changes in fluorescence intensity of TE671 cells upon addition of nAChR agonists epibatidine (10 µM, dark green), ACh (10 µM, blue), and nicotine (11 µM, light green), as well as assay buffer only (grey), after 20 s baseline recording. (B) Concentration-response plots showing the changes in the peak fluorescence intensity after addition of serial dilutions of epibatidine (dark green), ACh (blue), and nicotine (light green). (C) Representative traces showing changes in fluorescence intensity of TE671 cells after 15 min pre-incubation with 100 nM of the isolated Lc-α-NTx α-BgTx (red) and the Sc-α-NTx sNTx1 (yellow), followed by addition of 10 µM ACh after 20 s baseline recording. Representative traces of 10 µM ACh control (blue) and assay buffer (grey) are also included. (D) Concentration-inhibition plots showing the inhibition of peak fluorescence intensity of the 10 µM ACh response after the pre-incubation of serial dilutions of α-BgTx (red) and sNTx1 (yellow). Representative traces showing the changes in fluorescence intensity of TE671 cells after a first addition of 30 µM ACh (E) or 300 µM nicotine (F) followed by second addition of either assay buffer or 5 µM α-BgTx (concentrations stated in (E) and (F) are initial concentrations before addition to well, all other panels state final concentrations after addition to well). Each data point in (B) and (D) represents the mean (±SD) of three independent experiments (n = 3), the data points constituting each trace in (E) and (F) represent the mean of four replicate wells on a single plate with a total of 3 traces representing three independent experiments (n = 3).

Fig. 3

Responses of TE671 nAChRs with membrane potential dye are consistent across a 96-well plate. Scatter plots of the fluorescent response of TE671 cells from wells of a 96-well plate pre-incubated with concentrations of α-BgTx that either give maximal inhibition (30 nM, MIN, red), a middle level of inhibition (3 nM, MID, yellow), or no inhibition (none, MAX, blue), followed by the addition of 10 µM ACh. Each condition was applied to alternating columns of three separate plates (n = 3). Each row of plots contains data generated from one plate. Each plate was recorded on a different day with cells of a different passage number and the assigned MIN, MID, and MAX columns were changed on each plate. Each data point is the resulting RFU value after calculating F_max-F_baseline and are plotted by column (A) or by row (B). (C) Summary table of the RFU mean, standard deviation (SD) and coefficient of variance (CV) for each control (MIN, MID, MAX) for each plate. The final column presents the RFU mean once normalised to the MIN and MAX controls.

Fig. 4

Crude snake venoms show antagonism on nAChRs expressed in TE671 cells. Serial dilutions of crude venom (66.7 – 0.00067 µg/mL) extracted from neurotoxic elapid snake species with geographical distributions covering different regions of the African continent were pre-incubated with TE671 cells for 15 min followed by the addition of 10 µM ACh to create concentration-inhibition plots for each venom (outer ring). Each data point represents the mean (±SD) of three independent experiments (n = 3). To the top right of each plot on the outer ring are maps of the African continent highlighting in red the geographical distribution of each species. Maps were generated using QGIS, based on the 2019 International Union for Conservation of Nature Red List of Threatened Species. The central plot compares the IC₅₀ values (±95% CI) obtained from mamba (purple) and cobra (orange) venoms and IC₅₀ values are displayed above images of snakes inset to the bottom left of each plot.

Fig. 5

Inhibition of venom or isolated α-NTxs by commercial antivenom, small molecules, monoclonal antibodies, and decoy receptors. α-NTx-inhibitors of various formats were co-incubated with concentrations of crude venom (0.67 µg/mL for D. polylepis and 6.67 µg/mL for N. haje) or isolated α-NTx (30 nM) that gave approximate maximal inhibition prior to application to TE671 cells. All data points represent the mean (±SD) of three independent experiments (n = 3) and are normalised to 10 µM ACh (100% signal) and ACh + venom/α-NTx (0% signal) controls with α-NTx-inhibitory activity represented by recovery towards the 100% ACh signal. Venom/α-NTx concentrations were constant at 6.67 and 0.67 µg/mL for N. haje and D. polylepis respectively. (A) Concentration-response curves showing TE671 ACh response after crude D. polylepis (purple) and N. haje (orange) venoms were co-incubated with serial dilutions of SAIMR polyvalent antivenom (solid lines, 333.3 – 1.4 µg/mL) and EchiTAbG (dotted lines, 1670.0 – 0.2 µg/mL). Only SAIMR polyvalent antivenom showed α-NTx-inhibiting activity with EC₅₀s of 7.8 µg/mL for D. polylepis and 146.1 µg/mL for N. haje. (B) Screening of a panel of rationally selected small molecules at 100 µM co-incubated with crude D. polylepis (purple) and N. haje (orange) venom. Each experiment included controls of assay buffer only (no ACh), 3 nM and 30 nM α-BgTx, and 1% DMSO as the drug vehicle control (DMSO). Only brucinic acid (NSC 121865) showed α-NTx-inhibiting activity after incubation with N. haje venom, but D. polylepis venom was not inhibited. (C) Serial dilutions of the ‘decoy receptor’ Ls-AChBP were pre-incubated with either 30 nM α-BgTx or 30 nM sNTx1 at molar ratios ranging from 156:1 – 0.0016:1 and dilutions of the mAbs 2551_01_A12 (A12) and 2554_01_D11 (D11) specific to Lc-α-NTXs, and 367_01_H01 (H01) specific to dendrotoxins were co-incubated with 30 nM α-BgTx at molar ratios of 4.37:1, 2.19:1 and 1.09:1. Inhibition of α-BgTx activity was observed after Ls-AChBP was co-incubated with α-BgTx at molar ratios of 156:1 – 1.56:1 but no inhibition of sNTx1 was observed after further dilution. Inhibition of α-BgTx activity was observed with only 2554_01_D11 and 2551_01_A12 mAbs, with 2554_01_D11 showing a greater level of α-NTx inhibition than 2551_01_A12. To ensure the α-NTx-inhibitors themselves had no effect on nAChR activation, controls of mAb only (2551_01_A12, 2554_01_D11, and 367_01_H01 alone) and Ls-AChBP only at the highest concentrations used for pre-incubation with α-NTxs were also included.