An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

Abstract

Among the agents classified as “Category A” by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

“…SNAPtide (List Biological Laboratories) is a fluorogenic reporter for the detection of BoNT/A proteolytic activity that has long been used to characterize BoNT-drug interactions or BoNT enzymatic activity [49][50][51][52]. This reporter consists of a short peptide sequence with an internal donor/acceptor FRET pair, with the acceptor acting as a quencher.…”

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…Thus, it is important that this anomaly is more thoroughly investigated. The importance of determining the optimum length of a fully active LcA is more evident from the fact that some active site inhibitors showed nanomolar k i (19) when assayed with a short version of LcA but displayed micromolar k i (20,21) when assayed in its full-length, 448-residue version. Additionally, active site peptide inhibitors bound the full-length LcA with higher affinity than its shorter, 1-425-residue versions (22).…”

Antibody Protection against Botulinum Neurotoxin Intoxication in Mice

“…Such knowledge will result in the design of better therapeutics and treatment regiments. Thus far, the mouse model of botulism has been used extensively for toxin detection, for toxin concentration standardization, and in the development of therapeutics (9,12).…”