Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.

Alon Ben David,Ada Barnea,Eran Diamant,Ran Zichel,Eyal Dor,Amram Torgeman,Arieh Schwartz

doi:10.3390/ijms22168577

Abstract

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

Highlights

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature [1]
BoNTs bind to specific receptors on motor neurons, and after endocytosis, the light chain (LC) translocates into the cell cytoplasm, where it cleaves one of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins depending on the BoNT serotype
The entrance of BoNT/A into neurons begins with the interaction between the HCfragment domain of the toxin and the synaptic vesicle protein synaptic vesicle protein 2 (SV2) on neuronal cells, with the highest affinity toward the fourth luminal loop of variant SV2C [21] (Figure 1A)

Summary

Introduction

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature [1]. These toxins are produced by the Gram-positive spore-forming bacterium Clostridium botulinum. BoNTs are ~150 kDa proteins produced by the bacterium as a single polypeptide and thereafter proteolytically nicked to form a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) that are connected together by a disulfide bridge. Cleavage of the SNARE protein prevents neurotransmitter release to muscle cells. This results in flaccid muscle paralysis and can lead to respiratory failure and eventually death [5]

Methods

Results

Conclusion