Botulinum toxin is the etiologic agent responsible for the disease botulism, which is characterized by peripheral neuromuscular blockade. Botulism is ordinarily encountered as a form of oral poisoning. The toxin is absorbed from the lumen of the gut to reach the general circulation and is then distributed to peripheral cholinergic nerve endings. However, there is a widespread presumption that botulinum toxin can also act as an inhalation poison, which would require that it be absorbed from the airway. Experiments have been done to show that both pure toxin and progenitor toxin (a complex with auxiliary proteins) are inhalation poisons. Interestingly, the data indicate that auxiliary proteins are not necessary to protect the toxin or to facilitate its absorption. When studied on rat primary alveolar epithelial cells or on immortalized human pulmonary adenocarcinoma (Calu-3) cells, botulinum toxin displayed both specific binding and transcytosis. The rate of transport was greater in the apical-to-basolateral direction than in the basolateral-to-apical direction. Transcytosis was energy dependent, and it was blocked by serotype-specific antibody. The results demonstrated that the holotoxin was not essential for the process of binding and transcytosis. Both in vivo and in vitro experiments showed that the heavy-chain component of the toxin was transported across epithelial monolayers, which indicates that the structural determinants governing binding and transcytosis are found in this fragment. The heavy chain was not toxic, and therefore it was tested for utility as an inhalation vaccine against the parent molecule. This fragment was shown to evoke complete protection against toxin doses of at least 10 4 times the 50% lethal dose.

A robust, high throughput, two-tiered assay for screening small molecule inhibitors against botulinum neurotoxin serotype A was developed and employed to screen 16,544 compounds. Thirty-four compounds were identified as potent hits employing the first-tier assay. Subsequently, nine were confirmed as actives by our second-tier confirmatory assay. Of these, one displayed potent inhibitory efficacy, possessing an IC50 = 16 μM (± 1.6 μM) in our in vitro assay. This inhibitor (0831–1035) is highly water-soluble, and possesses an IC50 = 47 μM (± 7.0 μM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 μM) suggesting that this inhibitor is a good candidate for further development as a therapeutic countermeasure to treat botulism resulting from botulinum neurotoxin serotype A intoxication. An enzyme kinetics study indicated that this inhibitor exhibits mixed non-competitive inhibition, with a KI = 9 μM.

All serotypes of botulinum toxin possess a disulfide bond that links the heavy chain and light chain components of the holotoxin. Experiments were done to assess the functional significance of this covalent bond, and the work was facilitated by use of mercurial compounds that modify residues in the vicinity of the catalytic site. The data indicated that reduction of the interchain disulfide bond had two major effects: 1) changing conformation or orientation of the two chains, which diminished toxicity against intact cells, and 2) loosening or relocating a heavy chain belt segment that encircles the light chain and occludes the catalytic site. Interestingly, disulfide bond reduction of all serotypes produced conformational changes that diminished toxicity against intact cells, but it produced conformational changes that led to exposure of the catalytic site in only three serotypes. For the other serotypes, the catalytic site was accessible even before disulfide bond reduction. Neither of the major structural effects was dependent upon separation of the heavy chain and light chain components of the toxin, nor were they dependent on toxin substrate. Depending on the initial state of the toxin molecule, the combination of disulfide bond reduction and treatment with a mercurial compound could abolish toxicity. Therefore, this combination of treatments was used to convert active toxin into a parenteral vaccine. Administration of the modified toxin evoked a substantial IgG response, and it produced complete protection against a large dose of native toxin.

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