C. botulinum Neurotoxin Type A Light chain [GST]

Botulinum toxin is an agent that is typically encountered in two settings: as an agent that can cause disease (e.g. botulism), and as an agent that can be used to treat disease (i.e., a variety of neurologic disorders). In both cases it would be advantageous to develop a sound understanding of the mechanisms by which antibodies neutralize the toxin. In the present study, recombinant antigens were used to generate antibodies against the carboxyterminal half of the toxin heavy chain (HC50), the entire toxin light chain (LC), and the HA17, HA35 and HA70 components of the progenitor toxin complex. These antibodies were then evaluated for their respective abilities to alter botulinum toxin-induced changes in locomotor behavior in mice. The botulinum toxin type A complex was shown to produce dose-dependent depression of locomotor behavior within the dose range of 0.3-0.7 mouse LD50 units. At a dose of 0.5 LD50, the toxin typically reduced running behavior by 90% or more, and full recovery was not observed for approximately 4 weeks. Mice that were actively or passively vaccinated against the HC50 polypeptide were resistant to toxin action, presumably because the antibodies occluded the toxin binding domain. Interestingly, mice that were actively or passively vaccinated against LC were also resistant to toxin action. This effect may have been due to steric hindrance of the binding process. There was no scenario in which anti-HA antibodies altered the effects of toxin on locomotor behavior. This absence of effect was likely due to the fact that HAs and neurotoxin in the progenitor toxin complex spontaneously dissociate in physiologic media. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure-activity relationships and a proposed mechanism of action for this novel inhibitor chemotype. (C) 2011 Elsevier Ltd. All rights reserved.