The Strange Case of the Botulinum Neurotoxin: Using Chemistry and Biology to Modulate the Most Deadly Poison
TL;DR: Recent 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.
Abstract: 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.
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TL;DR: Recent findings of potential clinical importance that will advance the understanding of the effects of neuromuscular blocking agents and neuromUScular monitoring and also the management of disorders of the neuromoscular system within anaesthesia and intensive care are focused on.
Abstract: The neuromuscular junction (NMJ) is structured and powered to transduce electrical activity from the distal nerve terminal of a motor neurone via the neuromuscular cleft to the post-junctional muscle membrane to ultimately generate muscle contraction. Our understanding of this complex function has expanded over many years, and the NMJ has served as a prototype for how different synapses operate in the peripheral and central nervous systems. The NMJ has a presynaptic part which is synonymous with the distal nerve ending, being responsible for neurotransmitter synthesis, packaging into vesicles, and subsequent vesicle transportation to active release sites where vesicle docking, fusion, and release of acetylcholine and other co-released transmitters finally take place. The synaptic cleft, filled with large molecular complexes that guarantee ultrastructural NMJ arrangement and signal transduction, allows for rapid diffusion and degradation of the neurotransmitter. The postsynaptic part consists of a folded muscle membrane into which nicotinic acetylcholine receptors (nAChRs) directly opposite the presynaptic active release sites are mounted and fixed by a cytoskeleton. This specialized postsynaptic region is closely associated with the perijunctional zone where a high density of sodium channels promote and amplify the signal in order to guarantee the propagation of the electrical activity to generate muscle contraction. The transduction process is maintained at load (i.e. high stimulus frequency) by a presynaptic mechanism allowing for sustained transmitter release over time at high demand. This positive feedback mechanism relies on neuronal nAChRs present on the distal nerve terminal, whereas the continuation of the transduction process at the postsynaptic part relies on the classical muscle type nAChR. In this review, we will focus on recent findings of potential clinical importance that will advance our understanding of the effects of neuromuscular blocking agents and neuromuscular monitoring and also our management of disorders of the neuromuscular system within anaesthesia and intensive care.
100 citations
TL;DR: In conclusion, botulinum toxin may cause serious adverse events, which are more common after its therapeutic use, but can also be noticed after its cosmetic use.
Abstract: Although botulinum toxin is generally considered safe, its widespread use and the constantly expanded indications raise safety issues. This study aimed to review the serious and long-term adverse events associated with the therapeutic and cosmetic use of botulinum toxin. Serious adverse events included dysphagia, respiratory compromise, generalized muscle weakness, marked bilateral ptosis, pseudoaneurysm of the frontal branch of the temporal artery, necrotizing fasciitis, sarcoidal granuloma, Fournier gangrene, and cervical kyphosis. Death was attributed to botulism or anaphylactic shock. In conclusion, botulinum toxin may cause serious adverse events, which are more common after its therapeutic use, but can also be noticed after its cosmetic use. Thorough knowledge of the anatomy of the treated muscles and of the pharmacology of the drug is imperative to avoid serious adverse events.
88 citations
TL;DR: Using a single molecule assay of BoNT serotypes A and E light chain translocation through the heavy chain channel in neurons, it is discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy.
Abstract: Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.
84 citations
Cites background from "The Strange Case of the Botulinum N..."
...The crystal structures of the 7 LC serotypes have been solved, some with small molecules coordinated in the active site (2, 3, 26–31), making small molecule interaction modeling a powerful tool (6)....
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TL;DR: A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid, reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination.
Abstract: A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid (I1) is disclosed. A detailed evaluation of chicoric acid’s mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination. Other components found in Echinacea, I3 and I4, were also inhibitors of the protease.
48 citations
TL;DR: Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy and inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic).
Abstract: Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and have been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective postexposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the Johns Hopkins Clinical Compound Library composed of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960s, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a Ki of 6.7 ± 0.7 μM. The inhibition profile...
45 citations
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TL;DR: The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicles or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesICLE-to-target specificity.
Abstract: The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.
3,190 citations
TL;DR: This work substituted the CDRs from the heavy-chain variable region of mouse antibody B1–8, which binds the hapten NP-cap, for the corresponding CDRs of a human myeloma protein, to determine whether the antigen-binding site could be transplanted from one framework to another by grafting theCDRs.
Abstract: The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.
2,685 citations
TL;DR: The results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of the results, seems to play a key part in neurotransmitterRelease.
Abstract: Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.
1,727 citations
TL;DR: People potentially exposed to botulinum toxin should be closely observed, and those with signs of botulism require prompt treatment with antitoxin and supportive care that may include assisted ventilation for weeks or months.
Abstract: ObjectiveThe Working Group on Civilian Biodefense has developed consensus-based
recommendations for measures to be taken by medical and public health professionals
if botulinum toxin is used as a biological weapon against a civilian population.ParticipantsThe working group included 23 representatives from academic, government,
and private institutions with expertise in public health, emergency management,
and clinical medicine.EvidenceThe primary authors (S.S.A. and R.S.) searched OLDMEDLINE and MEDLINE
(1960–March 1999) and their professional collections for literature
concerning use of botulinum toxin as a bioweapon. The literature was reviewed,
and opinions were sought from the working group and other experts on diagnosis
and management of botulism. Additional MEDLINE searches were conducted through
April 2000 during the review and revisions of the consensus statement.Consensus ProcessThe first draft of the working group's consensus statement was a synthesis
of information obtained in the formal evidence-gathering process. The working
group convened to review the first draft in May 1999. Working group members
reviewed subsequent drafts and suggested additional revisions. The final statement
incorporates all relevant evidence obtained in the literature search in conjunction
with final consensus recommendations supported by all working group members.ConclusionsAn aerosolized or foodborne botulinum toxin weapon would cause acute
symmetric, descending flaccid paralysis with prominent bulbar palsies such
as diplopia, dysarthria, dysphonia, and dysphagia that would typically present
12 to 72 hours after exposure. Effective response to a deliberate release
of botulinum toxin will depend on timely clinical diagnosis, case reporting,
and epidemiological investigation. Persons potentially exposed to botulinum
toxin should be closely observed, and those with signs of botulism require
prompt treatment with antitoxin and supportive care that may include assisted
ventilation for weeks or months. Treatment with antitoxin should not be delayed
for microbiological testing.
1,659 citations
TL;DR: In hippocampal neurons, a Ca-activated K channel which may be responsible for the slow AHP has been identified in single channel recordings, however, this channel has unique properties and it is not blocked by Apamin, suggesting that more than one subclass of Ca- activated K channel may mediate slow AHPs.
1,311 citations