Concerns regarding botulinum neurotoxins (BoNTs) as biowarfare and bioterrorist agents have generated interest in developing medical countermeasures for protection against the neurotoxins. Efforts have focused on improvements in the available vaccines and antisera and de novo discovery of pharmacological inhibitors of toxin action. This chapter reviews the various approaches taken to develop next-generation vaccines, antitoxins and pharmacological treatments against intoxication by the BoNTs. The historical progression, current status and future trends are described.

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as "belt," that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that K(m) for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (k(cat)) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo.

ABSTRACT An integrated strategy that combined in silico screening and tiered biochemical assays (enzymatic, in vitro, and ex vivo) was used to identify and characterize effective small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A (BoNT/A). Virtual screening was initially performed by computationally docking compounds of the National Cancer Institute (NCI) database into the active site of BoNT/A light chain (LC). A total of 100 high-scoring compounds were evaluated in a high-performance liquid chromatography (HPLC)-based protease assay using recombinant full-length BoNT/A LC. Seven compounds that significantly inhibited the BoNT/A protease activity were selected. Database search queries of the best candidate hit [7-((4-nitro-anilino)(phenyl)methyl)-8-quinolinol (NSC 1010)] were performed to mine its nontoxic analogs. Fifty-five analogs of NSC 1010 were synthesized and examined by the HPLC-based assay. Of these, five quinolinol derivatives that potently inhibited both full-length BoNT/A LC and truncated BoNT/A LC (residues 1 to 425) were selected for further inhibition studies in neuroblastoma (N2a) cell-based and tissue-based mouse phrenic nerve hemidiaphragm assays. Consistent with enzymatic assays, in vitro and ex vivo studies revealed that these five quinolinol-based analogs effectively neutralized BoNT/A toxicity, with CB 7969312 exhibiting ex vivo protection at 0.5 μM. To date, this is the most potent BoNT/A small-molecule inhibitor that showed activity in an ex vivo assay. The reduced toxicity and high potency demonstrated by these five compounds at the biochemical, cellular, and tissue levels are distinctive among the BoNT/A small-molecule inhibitors reported thus far. This study demonstrates the utility of a multidisciplinary approach (in silico screening coupled with biochemical testing) for identifying promising small-molecule BoNT/A inhibitors.

A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50 kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS-polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8 kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research.

Botulinum neurotoxin (BoNT), the most lethal substance known, consists of a heavy chain (HC) and a light chain (LC). The LC specifically cleaves and inactivates the SNARE proteins, blocks neuronal exocytosis, and results in flaccid paralysis. The occurrence of multiple serotypes (A-G) presents notable challenges to the development of therapeutics using antibodies and peptide inhibitors. We initiated a structure-based approach to the identification of small molecule inhibitors of the proteolytic activity of BoNT. HPLC-based assays revealed a number of compounds that reduced the activity of LC-A or LC-B with serotype specificity, and several inhibited LCs of both serotypes. The apparent binding constants of the inhibitors to recombinant LC-A and LC-B were determined using fluorescence titration. Molecular modeling of the inhibitor-LC complexes revealed putative interactions of these compounds with the active sites of LC-A and LC-B. Further development of these lead inhibitors may provide effective therapeutics against botulism.

We investigated the inhibitory effect of stinging nettle leaf extract on the protease activity of botulinum neurotoxin type A and B light chains. The nettle leaf infusion was fractionated and HPLC-based enzymatic assays were performed to determine the capacity of each fraction to inhibit the protease activity of botulinum neurotoxin type A and B light chains. Assay results demonstrated that a water-soluble fraction obtained from the nettle leaf infusion inhibited type A, but did not inhibit type B light chain protease activity. The inhibition mode of water soluble fraction against protease activity of type A light chain was analyzed and found to be a non-competitive.