Covalent Modifiers of Botulinum Neurotoxin Counteract Toxin Persistence.

Botulinum neurotoxins (BoNTs) are the most potent toxins known to man and a significant threat as weapons of bioterrorism. BoNTs contain a metalloprotease domain that blocks neurotransmitter release in nerve terminals, resulting in a descending, flaccid paralysis with a 5-10% mortality rate. Existing treatment options cannot access or neutralize the toxin following its endocytosis, so there is a clear need to develop novel therapies. Numerous substrate-based and zinc-chelating small-molecule inhibitors have been reported; however, none have progressed to the clinic. This is likely due to the difficulty that reversible inhibitors have in achieving sustained inhibition of the toxin, which has a half-life of months in vivo. An alternative strategy for mitigating BoNT persistence is covalent, irreversible inhibition of toxin function. However, few examples of covalent BoNT inhibitors have been reported. Here, we describe a competition-based screen to identify covalent modifiers of the conserved active-site-adjacent cysteine C165 in the BoNT/A serotype. We found that compounds containing cysteine-reactive electrophiles designed to target cysteine proteases failed to bind C165 while selenide compounds were efficient covalent binders of this cysteine. Importantly, covalent modification at C165 resulted in sustained, irreversible inhibition of BoNT/A protease activity. Covalent selenide inhibitors were nontoxic and protective in a neuronal assay of intoxication, making them promising new scaffolds for the study of the BoNT/A toxin as well as for the design of novel therapy agents.

High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 µM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.

Recent developments with metalloprotease inhibitor class of drug candidates for botulinum neurotoxins.

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

Cyclophilin-facilitated membrane translocation as pharmacological target to prevent intoxication of mammalian cells by binary clostridial actin ADP-ribosylated toxins.

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 µM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.

Structure-based drug discovery for botulinum neurotoxins.

Clostridium botulinum neurotoxin is the most poisonous substance known to humans. It is a potential biowarfare threat and a public health hazard. The only therapeutics available is antibody treatment which will not be effective for post-exposure therapy. There are no drugs available for post-intoxication treatment. Accordingly, it is imperative to develop effective drugs to counter botulism. Available structural information on botulinum neurotoxins both alone and in complex with their substrates offers an efficient method for designing structure-based drugs to treat botulism.

Botulinum neurotoxins B and E translocate at different rates and exhibit divergent responses to GT1b and low pH.

Botulinum neurotoxins (BoNTs, serotypes A-G) are the most deadly substances known. Here, we investigated how BoNT/E, a serotype that causes human botulism, translocates into the cytosol of neurons. Analogous to BoNT/B, BoNT/E required binding of the coreceptor, GT1b, to undergo significant secondary structural changes and transform into a hydrophobic protein at low pH. These data indicate that both serotypes act as coincidence detectors for both GT1b and low pH, to undergo translocation. However, BoNT/E translocated much more rapidly than BoNT/B. Also, BoNT/E required only GT1b, and not low pH, to oligomerize, whereas BoNT/B required both. In further contrast to the case of BoNT/B, low pH alone altered the secondary structure of BoNT/E to some degree and resulted in its premature inactivation. Hence, comparison of two BoNT serotypes revealed that these agents exhibit both convergent and divergent responses to receptor interactions, and pH, in the translocation pathway.

Peptide inhibitors targeting Clostridium difficile toxins A and B.

Clostridium difficile causes severe hospital-acquired antibiotic-associated diarrhea due to the activity of two large protein toxins. Current treatments suffer from a high relapse rate and are generating resistant strains; thus new methods of dealing with these infections that target the virulence factors directly are of interest. Phage display was used to identify peptides that bind to the catalytic domain of C. difficile Toxin A. Library screening and subsequent quantitative binding and inhibition studies showed that several of these peptides are potent inhibitors. Fragment-based computational docking of these peptides elucidated the binding modes within the active site. These antitoxin peptides may serve as potential lead compounds to further engineer peptidomimetic inhibitors of the clostridial toxins.

Neonatal immune response of Brazilian beef cattle to vaccination with clostridium botulinum toxoids types C and D by indirect ELISA

Types C and D strains of Clostridium botulinum are commonly related to avian and mammalian botulism. Although there are numerous vaccine recommendations, little research has been conducted to indicate the real effectiveness of vaccine timing or the ideal immunization protocol for young beef calves. Four commercially available vaccines, two bivalent (Clostridium botulinum types C and D; vaccines 1 and 2) and two polyvalent (all Clostridium spp. including Clostridium botulinum types C and D; vaccines 3 and 4), that are currently used in Brazilian herds, were tested in order to verify the maternal immune response. One hundred cows, divided into four vaccinated groups and one unvaccinated group, were given a two-dose subcutaneous immunization, at day zero, followed by a second dose given at 42 days post-vaccination, which corresponded to 40 days before birth. Serum samples (n = 75) were collected only from healthy neonatal calves at 0, 7, 45 and 90 days post-calving (DPC) and subjected to indirect ELISA using the purified C and D holotoxins as capture antigens. The serological profile showed that all vaccines were able to induce a satisfactory neonatal immune response to both holotoxins at 7 DPC. However, at 45 and 90 DPC, a significant reduction (p < 0.05) was observed in the antibody level against C and D holotoxins in all tested vaccines. Neonatal immunization in calves is compromised by significant levels of maternal antibodies so that the necessity of planning a calf vaccination program involves assessment of disease risks at the production site. Finally, our findings represent the first demonstration of maternal immunity transferred to neonatal beef calves, including immunity levels after vaccination against Clostridium botulinum toxoids C and D.

Synthetic substrate for application in both high and low throughput assays for botulinum neurotoxin B protease inhibitors.

A FRET peptide substrate was synthesized and evaluated for enzymatic cleavage by the BoNT/B light chain protease. The FRET substrate was found to be useful in both a high throughput assay to uncover initial 'hits' and a low throughput HPLC assay to determine kinetic parameters and modes of inhibition.

Antibody protection against botulinum neurotoxin intoxication in mice.

Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.

Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F.

Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison.

In the classic novella "The Strange Case of Dr. Jekyll and Mr. Hyde", Robert Louis Stevenson paints a stark picture of the duality of good and evil within a single man. Botulinum neurotoxin (BoNT), the most potent known toxin, possesses an analogous dichotomous nature: It shows a pronounced morbidity and mortality, but it is used with great effect in much lower doses in a wide range of clinical scenarios. Recently, tremendous strides have been made in the basic understanding of the structure and function of BoNT, which have translated into widespread efforts towards the discovery of biomacromolecules and small molecules that specifically modulate BoNT activity. Particular emphasis has been placed on the identification of inhibitors that can counteract BoNT exposure in the event of a bioterrorist attack. This Review summarizes the current advances in the development of therapeutics, including vaccines, peptides, and small-molecule inhibitors, for the prevention and treatment of botulism.

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

BACKGROUND: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

A natural human IgM antibody that neutralizes botulinum neurotoxin in vivo.

Affinity-matured human antibodies have demonstrated efficacy as countermeasures for exposure to botulinum neurotoxin (BoNT), which is the cause of the disease botulism category A select bioterror agent. Little is known, however, about the potential role of natural (un-mutated) antibodies in the protective immune response to BoNT. Here we describe the cloning of two human IgM antibodies that bind serotype A BoNT. Both are un-mutated IgM antibodies, consistent with an origin in naive B cells. One of the antibodies is able to fully neutralize a lethal dose of serotype A BoNT in vivo. These results suggest that the natural human antibody repertoire may play a role in protection from exposure to biological toxins.

Use of a recombinant fluorescent substrate with cleavage sites for all botulinum neurotoxins in high-throughput screening of natural product extracts for inhibitors of serotypes A, B, and E.

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.

Strategies to design inhibitors of Clostridium botulinum neurotoxins.

Botulinum neurotoxins (BoNTs), produced by spore-forming anaerobic Clostridium botulinum, are the most toxic substances known. They cause the life-threatening disease botulism, characterized by flaccid muscle paralysis. While the natural cases of botulism are rare, due to their extreme toxicity and easy production, BoNTs have become potential biowarfare agents, and create maximum fear among populations concerned with bioterror agents. The only available antidote against BoNTs is equine antitoxin. Equine antitoxin can only target the toxins at extracellular level, and can not reverse the paralysis caused by botulism. In addition, equine antibody can cause severe hypersensitivity reactions, and is limited to be used for prophylaxis treatment. BoNTs are large proteins with three distinct domains, the binding domain, the translocation domain, and the enzymatic domain with highly specific endopeptidase activity to cleave the proteins involved the neurotransmitter release. Targeting any of these domains can inhibit the functions of BoNT. Humanized monoclonal antibodies, small peptides and peptide mimetics, receptor mimics, and small molecules targeting the endopeptidase activity have emerged as potential new inhibitors against BoNTs. With the structure of BoNT resolved, molecular modeling and rational design of potent antidotes against botulism is on the horizon. An area that has not been explored for designing the antidotes against botulism is aptamers, which have been successfully developed as therapeutics in several areas. This review will focus on some of these new strategies to design effective antidotes against botulism. The strategies reviewed in this article can be easily applied to design inhibitors for other bacterial toxins.

Human milk SIgA binds to botulinum type B 16S toxin and limits toxin adherence on T84 cells.

Botulinum neurotoxin produced by Clostridium botulinum type B is in the form of a complex of 12S and 16S toxins. Food-borne botulism is caused by these complex toxins which are ingested orally and absorbed from the digestive tract. Here, we show that the human milk SIgA binds to the type B16S toxin. The binding of SIgA to 16S toxin and HA was inhibited by carbohydrates such as galactose, suggesting that the interaction of carbohydrate side chain of the SIgA with the HA of the 16S toxin is important for SIgA-16S complex formation. We also demonstrate that SIgA inhibits the attachment of 16S toxin to intestinal epithelial cells. These data suggest that the interaction of antigen nonspecific SIgA with 16S toxin has a large influence on the absorption of 16S toxin from the intestinal epithelium, and that SIgA may provide insight into developing a therapeutic agent for type B food-borne botulism.

[Biological effects of toosendanin, an active ingredient of herbal vermifuge in Chinese traditional medicine].

The fact that the fruit and bark of plant belonging to family Melia could be used as digestive tract-parasiticide and agricultural insecticide was recorded about two thousand years ago in ancient China. Toosendanin (TSN, C30H38O11, FW=574), a triterpenoid derivative, was extracted from the bark of Melia toosendan Sieb. et Zucc. by Chinese scientists in 1950os and used as an ascarifuge in China instead of imported sendanin. Studies have demonstrated that TSN possesses special biological actions as well as considerable various values in scientific research, clinic medicine and agriculture. The first is that by interfering with neurotransmitter release by causing an initial facilitation, TSN eventually blocks synaptic transmission at both the neuromuscular junction and central synapses. The action might result from TSN-induced Ca(2+)-sensitivity change and final elimination of transmitter release machinery. The second is that despite sharing many similar actions with botulinum neurotoxin (BoNT) on blocking neuromuscular transmission, TSN has a markedly antibotulismic action in vivo and in vitro: TSN-treatment saves the botulism mice or monkeys from death; TSN-incubation in vitro or TSN-injection in vivo endows neuromuscular junction with a high tolerance to BoNT. Studies suggest that the antibotulismic action is achieved by preventing BoNT from approaching its enzymatic substrate, SNARE protein. The third, in recent years, it is also observed that TSN can induce differentiation and apoptosis in several cell lines, and suppress proliferation of various human cancer cells. The TSN-induced differentiation is Ca(2+)-dependent and the mitochondria-dependent apoptosis pathway is involved in the TSN-induced apoptosis. The fourth is that TSN inhibits various K(+) channels and selectively facilitates Ca(2+) current through L-type Ca(2+) channels and hence elevates [Ca(2+)](i). The TSN-induced [Ca(2+)](i) increase and overload could be responsible for the TSN-induced biphasic effect on neurotransmitter release, cell differentiation, apoptosis as well as the cytotoxicity of TSN.

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Botulinum neurotoxin serotype F: identification of substrate recognition requirements and development of inhibitors with low nanomolar affinity.

Botulinum neurotoxins (BoNTs A-G) are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in neurotransmitter release. In view of the extreme toxicities of these molecules, their applications in human medicine, and potential for misuse, it is of considerable importance to elucidate the mechanisms underlying substrate recognition and to develop inhibitors, with the ultimate goal of obtaining anti-botulinum drugs. We synthesized peptides based on vesicle-associated membrane protein (VAMP) to investigate the substrate requirements of BoNT F, which cleaves VAMP between residues Q58 and K59. The minimum substrate was a peptide containing VAMP residues 32-65, which includes only one of the two VAMP structural motifs thought to be required for botulinum substrate recognition. BoNT F exhibited a strict requirement for residues D57 (P(2)), K59 (P(1)'), and L60 (P(2)'), but peptides containing substitutions for R56 (P(3)), Q58 (P(1)), and S61 (P(3)') were cleaved. Therefore, the P(2), P(1)', and P(2)' residues of VAMP are of paramount importance for BoNT F substrate recognition near the scissile bond. K(i) values of uncleavable analogues were similar to K(m) values of the substrate, suggesting that substrate discrimination occurs at the cleavage step, not at the initial binding step. We then synthesized inhibitors of BoNT F that incorporated d-cysteine in place of glutamine 58, exhibited K(i) values of 1-2 nM, and required binding groups on the N-terminal but not the C-terminal side of the zinc ligand. The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B.