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.

The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity.

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 IC(50)=16 microM (+/-1.6 microM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC(50)=47 microM (+/-7.0 microM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 microM), 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 K(I)=9 microM.

Progress toward development of an inhalation vaccine against botulinum toxin.

The looming threat of bioterrorism has enhanced interest in the development of vaccines against agents such as botulinum toxin. This in turn has stimulated efforts to create vaccines that are effective by the oral and inhalation routes. Recently, considerable progress has been made in creating an inhalation vaccine against botulism. This work stems from the discovery that a polypeptide that represents a third of the toxin molecule retains the ability to be adsorbed from the airway and to evoke an immune response but retains none of the adverse effects of the native toxin. Interestingly, this polypeptide can also serve as a carrier molecule in the creation of inhalation vaccines against other pathogens.

The role of the interchain disulfide bond in governing the pharmacological actions of botulinum toxin.

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.

Immunologic characterization of spasmodic dysphonia patients who develop resistance to botulinum toxin.

The immune status of six spasmodic dysphonia patients who became resistant to botulinum toxin was compared to that of a series of patients who remained responsive. The two groups were similar in terms of age, sex, and cumulative dose of toxin. Five of the resistant patients had a significant titer of anti-botulinum toxin IgG antibodies, as determined by enzyme-linked immunosorbent assay (ELISA). These same five resistant patients had a circulating titer of anti-heavy chain antibodies, but only three of these patients had a circulating titer of anti-light chain antibodies, as determined by Western blotting. By contrast, none of the responsive patients had antibodies against the holotoxin or its two chains. Interestingly, two of the resistant patients also had a low circulating titer of anti-botulinum toxin IgA antibodies. None of the responsive patients was IgA-positive. The cumulative dose of botulinum toxin administered to resistant patients was lower than that customarily associated with emergence of immunity in dystonia patients.

Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component.

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.

Botulinum toxin type B for treatment of spasmodic dysphonia: a case report.

Although treatment with botulinum toxin type A (BTXA) has become the standard of care for most patients with laryngeal dystonia, its use is limited by the development of resistance to the toxin in some patients. Botulinum toxin type B (BTXB) has been found to be safe and effective in the treatment of cervical dystonia, but it has not been used previously to treat spasmodic dysphonia. Our experience with BTXB in a patient who developed resistance to BTXA suggests that BTXB may be safe and effective for the treatment of laryngeal dystonia, as well.

Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance.

To understand the mechanisms of antiviral drug resistance and to have a system to examine the cytotoxicity of herpes simplex virus type 1 (HSV-1) inhibitors that are thymidine kinase (TK)-dependent, we have constructed a plasmid pFTK1 by inserting a DNA fragment containing the TK gene of HSV-1 strain F into the eukaryotic expression vector pcDNA3.1/His A. TK-deficient 143B cells were transfected with this vector and neomycin-resistant cells were selected. Cell survival in HAT medium and TK activity of the cell lysates were examined to ascertain HSV-1 TK expression. A cell line expressing the viral TK gene, FTK143B (FTK), was established and used for characterization of two laboratory-derived TK-deficient drug-resistant HSV-1 mutants of strain F. The antiviral activities of several drugs, mostly nucleoside analogues, were compared in the Vero, 143B and FTK cell culture systems. We showed that both mutant viruses lost their resistance to acyclovir and to other HSV-1 TK-dependent compounds in FTK cells but not in Vero and 143B cells. Significantly increased cytotoxicity of ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine was also observed in the FTK cells. This HSV-1 TK gene-transfected cell model is a useful tool to rapidly determine HSV-1 drug resistance at the viral TK level.