Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active-site inhibitors of BoNT/A light chain using an integrated multi-assay approach. ABS 252 showed inhibitory activity in enzymatic, cell-based and muscle activity assays, and importantly, produced a marked delay in time-to-death in mice. The results suggest that a multi-assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates.

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

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 botulinum neurotoxin A in a spiked milk sample with subtype identification through toxin proteomics.

Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT in buffer. This method can rapidly determine the presence of BoNT in a sample and differentiate the toxin type of BoNT present but does not yield additional information about the subtype. We now describe here the application of Endopep-MS to detect BoNT A in a spiked milk sample. This work also describes subtype identification achieved through mass spectrometric analysis of the protein toxin itself and does not require the presence of DNA from the toxin-producing bacteria. Tryptic digests of A1 and A2 subtypes of BoNT were analyzed by mass spectrometry, and peptides unique to either the A1 or A2 subtype were subjected to tandem mass spectrometry analysis to confirm their identities. Finally, subtype identification through mass spectrometric analysis was performed on BoNT A isolated from spiked milk. In its entirety, this method would allow for analysis of BoNT with toxin type identification in a few hours and subtype identification within 24 h.

Arginine Promotes Toxin Formation in Cheddar Cheese by Clostridium botulinum.

The production of botulinal toxin by a mixture of spores of Clostridium botulinum types A and B was evaluated in Cheddar cheese supplemented with L-arginine (1% wt/wt) and containing one of three levels of sodium chloride (0, 0.9, or 1.8%). Botulinal toxin was formed in cheeses containing an increased level of L-arginine (1%) and reduced levels of sodium chloride (0 or 0.9%). No toxin was formed in Cheddar with arginine and 1.8% salt or in any of the cheeses not supplemented with arginine. The pH increased from 5.05-5.2 to 5.7-6.0 in the cheeses with increased arginine, but the pH change alone did not permit growth of C. botulinum . Metabolism of arginine may also have promoted the synthesis of compatible metabolites for salt resistance. The results indicate that an important factor supporting growth of C. botulinum in cheese is the availability of L-arginine.

Evaluation of the Botulism Hazard from Vacuum-packaged Enoki Mushrooms ( Flammulina velutipes ).

Flammulina velutipes , commonly known as the enoki or winter mushroom, is cultured aseptically in Japan and the United States and is vacuum-packaged in polyethylene film for retail sale. Because of the known potential hazard of botulism from agaric mushrooms that are packaged in plastic films, the safety of vacuum-packaged enokis was evaluated. Botulinal toxin was not detected by mouse assay in 148 packages from 14 independent lots when a rich medium containing trypticase-peptone-glucose-yeast extract, was added aseptically to the packages which were heat-shocked (60 or 80°C for 20 min) and incubated at 30 or 37°C. Botulinal toxin was produced when spores were added to the packages, but spoilage was evident prior to toxin formation. Toxin was not formed when the inoculated packages were kept refrigerated (6°C). The results indicate that fresh vacuum-packaged enoki mushrooms do not present a botulism hazard when cultured aseptically and stored refrigerated.