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

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.

Centaur antibodies: Engineered chimeric equine-human recombinant antibodies

“…All recombinant antigens contained 6xhis tag. The proteins were expressed and purified as described previously (18)(19)(20).…”

Biological and Immunological Characterization of a Functional L-HN Derivative of Botulinum Neurotoxin Serotype F

“…BoNTs are the most toxic biotoxins, which can cause nerve paralysis syndrome in mammals and other vertebrates, and are classified as class A biological warfare agents by the US centers for disease control and prevention (CDC) [ 3 , 4 ]. According to the neutralization effects of specific antisera, BoNTs can be divided into seven traditional serotypes, A–G, and novel botulinum neurotoxins, including BoNT/H (previously considered to be a chimeric neurotoxin of BoNT/F and BoNT/A) and BoNT/X [ 5 , 6 , 7 , 8 , 9 ]. Among them, botulinum neurotoxin serotypes A, B, E, and F cause human poisoning, while BoNT/C and D cause neurotoxicity in animals [ 10 ].…”

Recent Advancements and Novel Approaches Contributing to the Present Arsenal of Prophylaxis and Treatment Strategies Against Category A Bacterial Biothreat Agents

“…Chauhan et al [ 51 ] in their study have shown their two 8-hydroxyquinoline candidate compounds: NSC1011 and NSC1014 to have remarkable extension in survival in mice models against BoNT/F challenge. Receptor binding inhibitor molecules like aurintricarboxylic acid (ATA) provided protection against BoNT A/E challenge [ 52 ]. These novel molecules and approaches hold promise towards a concluding therapeutic for BoNT spectrum pathogenesis (Table 4 ).…”