Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Timothy Lynagh,Brian Lohse,Martin Dufva,Daniel Kümmel,Jónas Johannesen,Bengt H Gless,Maria Meyhoff-Madsen,Stephan Kiontke,Kanchan Devkota,Sabrina Kattelmann,Christian A Olsen,Andreas H Laustsen,Stephan Alexander Pless,Anders Christiansen

doi:10.1021/acs.jmedchem.0c01202

Abstract

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure–function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Highlights

Snakebite envenoming kills or maims hundreds of thousands of people every year,[1,2] and loss of livestock is emerging as a substantial problem.[3]
We heterologously expressed nicotinic α7-homomeric nicotinic acetylcholine receptors (nAChRs) in Xenopus laevis oocytes and measured acetylcholine-gated currents in response to acetylcholine alone, in the presence of αCbtx, or in the presence of α-Cbtx preincubated with the identified peptides
Α-Cbtx alone (40 nM) inhibited nAChR responses to ∼10% compared to the control level, and washout of this effect was minimal, with subsequent responses to ACh in the absence of α-Cbtx recovering to only 20% (Figure 1A,B)

Summary

Introduction

Snakebite envenoming kills or maims hundreds of thousands of people every year,[1,2] and loss of livestock is emerging as a substantial problem.[3]. In the case of elapid snakes, such as cobras and kraits, threefinger α-neurotoxins such as α-cobratoxin (α-Cbtx) and αbungarotoxin (α-Bgtx) are the most prominent and lethal venom components.[9−11] α-Cbtx and α-Bgtx potently inhibit postsynaptic nicotinic acetylcholine receptors (nAChRs), which depresses neuromuscular signaling, causing paralysis and loss of respiration.[12] The effectiveness of conventional antivenoms is limited by immunogenicity, abundance of α-neurotoxin antibodies in the immunized animal at the time of production,[13] and which type of snake inflicts the bite. Newer antibody-based strategies have emerged, with a focus on smaller, less immunogenic, and venom-specific antivenoms. These showed promising neutralization of dendrotoxins in mice, some improvement is needed in neutralizing α-neurotoxins

Results

Discussion

Conclusion