Bimodal modulation of the botulinum neurotoxin protein-conducting channel.

Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.

A neuronal cell-based botulinum neurotoxin assay for highly sensitive and specific detection of neutralizing serum antibodies.

Clostridium botulinum neurotoxin (BoNT) serotypes A and B are widely used as pharmaceuticals to treat various neurological disorders and in cosmetic applications. The major adverse effect of these treatments has been resistance to treatment after multiple injections. Currently, patients receiving BoNT therapies and patients enrolled in clinical trials for new applications and/or new formulations of BoNTs are not routinely monitored for the formation of neutralizing antibodies, since no assay other than the mouse protection procedure is commercially available that reliably tests for the presence of such antibodies. This report presents a highly sensitive and specific neuronal cell-based assay that provides sensitive and specific detection of neutralizing antibodies to BoNT/A.

Synthesis of soluble multivalent glycoconjugates that target the Hc region of botulinum neurotoxin A.

The design, synthesis, and initial inhibitory studies of di- and tetravalent glycoconjugates that target the heavy chain of botulinum neurotoxin A are reported.

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.