Botox reversal: Taming botulinum toxin to deliver therapeutics

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While rare, botulism can cause paralysis and is potentially fatal. It is caused by nerve-damaging toxins produced by Clostridium botulinum – the most potent toxins known.

These toxins are often found in contaminated food (home canning being a major culprit). Infants can also develop botulism from ingesting C. botulinum spores in honey, soil, or dust; the bacterium then colonizes their intestines and produces the toxin.

Once paralysis develops, there is no way to reverse it, other than waiting for the toxins to wear off.

People with serious cases of botulism may need to be maintained on ventilators for weeks or months. But a new treatment approach and delivery vehicle, described today in Science Translational Medicine, could change that.

“There are anti-toxins, but these only work before the toxins enter the motor neurons,” says Min Dong, Ph.D., a researcher in Boston Children’s Hospital’s Department of Urology and corresponding author on the paper.

“What we have developed is the first therapy that can eliminate toxins after they get inside neurons.”

If proven in humans, the approach would represent a breakthrough in treating botulism. In mice, the treatment successfully got inside neurons, reversed muscle paralysis within hours, and enabled mice to withstand doses of botulinum toxin that would otherwise be lethal.

Letting a toxin lead the way

Dong and his colleagues needed to surmount two technical barriers that have prevented the botulism from being treated effectively in the past. Intriguingly, their solution lay in botulinum toxin itself.

“One barrier to treatment has been getting across the cell membrane, which is difficult for protein drugs,” explains Shin-Ichiro Miyashita, Ph.D., a postdoctoral fellow in Dong’s lab and first author on the paper.

“The other is targeting specific cell types, and in this case specificity toward motor neurons and nerve terminals. We took advantage of the fact that botulinum neurotoxins target motor neurons naturally and efficiently, and can deliver a protein cargo across cell membranes.”

The treatment is therefore two-pronged. A botulinum toxin (detoxified through introduced mutations) is the delivery vehicle. The cargo – i.e., the active drug – is a mini-antibody derived from the antibodies of camels, developed by collaborator Charles Shoemaker, Ph.D., at Tufts University.

The team showed that two of these so-called nanobodies can be delivered in tandem into neurons, neutralizing botulinum toxins type A and B at one go.

But there was one more problem to solve.

“This idea and approach had been attempted, but it was difficult to completely get rid of toxicity,” says Dong, “until we identified a new toxin, botulinum neurotoxin X, in 2017. Unlike other botulinum toxins, this new toxin shows no toxicity after we introduce mutations, and serves as a safe delivery tool.”

Botox reversal

Besides botulism, Dong thinks the new treatment could be useful as a “botox reversal” agent. Botox injections, using tiny quantities of the type A botulinum toxin, can safely treat wrinkles and many other medical conditions like neck spasms, excessive sweating, or overactive bladder. However, when the injection goes awry, botox can cause unwanted muscle paralysis as a side effect, and patients have to live with the paralysis for months.

“We can potentially inject our therapeutic protein and get rid of botox in neurons and paralysis within a few hours,” Dong says.

A general delivery platform for neuroactive drugs?

The toxin-guided approach may offer a platform for getting biologic drugs into neurons to treat other disorders, Dong believes. Currently, most biologic drugs act only on cell-surface targets and cannot get into the cell’s interior.

“We provide a protein-based drug delivery platform that achieves highly specific targeting of neurons and efficient penetration of cell membranes,” Dong says.

“Combined with nanobodies, which can be developed fairly readily against any protein of interest, this platform can be used to develop therapeutics that modulate proteins and biological processes inside neurons. Its modular nature even allows us to target cell types other than neurons by switching the cell-targeting domain. This could present a general approach for precision drug delivery into cells.”


STRUCTURE AND MODE OF ACTION OF CLOSTRIDIAL NEUROTOXINS

Botulinum neurotoxins (BoNTs), serotypes A through H, produced by Clostridium botulinum, and the related tetanus toxin (TeNT) produced by C. tetani, are one of the groups of toxins, for which tremendous progress has been made in the past couple decades toward our understanding of their structure and function.

BoNT serotypes A and B, and to a lesser extent E and F (and now H), are associated with human botulism cases, while serotypes C and D are more prevalent in bird and animal botulism [14]. These toxins are produced as single-chain proteins of approximately 150 kDa and share 3–30% primary amino acid similarity among each other [14–15]. Despite the sequence differences among the serotypes, all these proteins share a common structure and mechanism of action and are considered the most potent toxins for humans.

BoNTs cause flaccid paralysis, while TeNT causes spastic paralysis. Some BoNTs (e.g., BoNT/A) are produced in bacteria as a protein complexed with stabilizing non-toxic neurotoxin-associated proteins [16], but the toxin alone is capable of intoxication [17]. BoNTs are activated by proteolytic cleavage to generate an N-terminal 450 amino acid fragment (light chain = LC, ~50 kDa) comprised of the catalytic zinc-dependent protease domain [18] and a C-terminal 800 amino acid fragment comprised of the translocation and receptor-binding domains (heavy chain = HC, ~100 kDa) [19]. The dichain is held together by a disulfide bond, which is reduced upon translocation into the cytosol of the neuronal cell.

The crystal structures are available for three of the holotoxins, BoNT/A [20], BoNT/B [21], and BoNT/E [22], as well as various domain fragments of these and other serotypes, alone or in complex with their ligands, substrates or receptors [23]. The crystal structure of BoNT/A (Fig. 1, PDB 3BTA) revealed three functional domains: a receptor-binding domain made up of two sub-domains, a translocation domain consisting of long α-helices and an unusual belt (green in Fig. 1) that wraps around the zinc-containing catalytic domain along the substrate-binding sites and occludes the active site pocket [20, 24].

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Fig. (1)
Structure of BoNT/A. Shown is a molecular graphic representation of the BoNT/A structure depicting the three functional domains, generated using Pymol (PDB 3BTA): N-terminal catalytic domain (LC), residues 1–431 (yellow); active site Zn2+ (red); belt region, residues 450–544 (green); membrane-translocation domain (TD), residues 545–870 (magenta); disulfide bond between Cys429, Cys453 (cyan); and a C-terminal ganglioside and protein co-receptor-binding domain (BD) consisting of two subdomains, residues 871–1091 (orange) and 1092–1295 (blue).

BoNTs bind to receptors on neurons at the presynaptic ending, undergo receptor-mediated endocytosis, and in the low pH of the endocytic vesicle are translocated into the cytosol of the neuron, where SNARE proteins are subjected to proteolysis by the released zinc-dependent protease domain [25–29]. SNARE stands for soluble N-ethylmaleimide-sensitive factor activating protein receptor.

SNARE proteins are a complex of proteins that are involved in membrane fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane at nerve endings and include the proteins SNAP25, VAMP/synaptobrevin, and syntaxin. Proposed models of BoNT intoxication are illustrated in Fig. (2), and involve the following steps: (Step 1) binding of the C-terminal portion (HC) of BoNT-HC to presynaptic membrane receptors, (Step 2) uptake into an intracellular vesicle, and (Steps 3–6) translocation of the catalytic light chain (BoNT-LC) into the cytosol via the N-terminal portion (HN) of BoNT-HC. Delivery of the catalytic BoNT-LC into the cytosol then leads to BoNT-LC-mediated proteolytic cleavage of serotype-specific SNARE-containing proteins involved in synaptic vesicle function.

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Fig. (2)
Proposed model of BoNT intoxication of motor neurons. Shown is a schematic diagram of the proposed mode of entry and translocation of the catalytic activity cargo into the cytosol of neuronal cells. In this model, the holoprotein is cleaved either by clostridial or cellular proteases into a 50-kDa catalytic domain (LC), which is a Zn2+-dependent protease that cleaves SNARE proteins involved in synaptic vesicle exocytosis, linked by a disulfide bond to a 100-kDa HC, which mediates the binding, entry and delivery of the LC (yellow) into the cytosol of neuronal cells, where the LC catalyzes the cleavage of its cognate SNARE-protein substrate(s) to block neurotransmitter release and cause paralysis. Step 1: The C-terminal binding domain (BD) of the HC comprised of two subdomains (orange and blue) bind to surface gangliosides and/or neuronal-specific protein co-receptors (Syt-I, Syt-II, SV2A, SV2B or SV2C). Step 2: The toxin-receptor complex triggers uptake into cells via receptor-mediated endocytosis. Step 3: Acidification of the endosomes induces a conformational change in the N-terminal membrane-translocation domain (TD, magenta) of the HC that leads to its interaction with the endosomal membrane. Steps 4–6: Two alternative paths leading to translocation of the LC across the endosomal membrane that are consistent with experimental findings are shown. In path 4a–6a, the HC remains intact and the low pH induces insertion into the vesicle membrane, where with the assistance of the BD the TD forms a channel and facilitates the unfolding and funneling of the LC through the channel into the cytosol, where the LC refolds and acts on its target substrate(s). In path 4b–6b, the BD dissociates from the TD and the TD alone facilitates the unfolding and translocation of the LC across the membrane and into the cytosol.

BoNT-LC-mediated cleavage of the SNARE proteins inhibits exocytosis of neurotransmitter-containing synaptic vesicles by preventing fusion of the synaptic vesicles with the plasma membrane and subsequent acetylcholine neurotransmitter release into the synapse. The molecular targets of BoNTs have been defined [27–32]. All BoNTs cleave their substrates at specific peptide bonds: BoNT serotypes B, D, F and G, as well as the related TeNT from C. tetani, cleave synaptobrevin (also called VAMP), a membrane protein found in small synaptic vesicles; BoNT serotypes A, C and E cleave SNAP25 at the presynaptic plasma membrane; and BoNT serotype C also cleaves syntaxin at the plasma membrane. The substrate specificity of the newly identified BoNT/H [30–31] has not yet been reported.

Current models propose that the host receptors for BoNTs and TeNT define their ultimate localization in the nerve cell [33]. Binding and endocytosis of clostridial neurotoxins is thought to occur via multiple membrane receptors: gangliosides that bind toxins with low affinity and/or neuron-specific protein co-receptors, the synaptic vesicle membrane protein synaptotagmin (Syt-I, Syt-II) and/or synaptic vesicle glycoproteins (SV2A, SV2B, and SV2C) [34].

These form high-affinity, high-specificity complexes [34], which cluster within arrays of presynaptic receptors at the peripheral nerve terminal [35]. Upon binding, the BoNTs enter cells via receptor-mediated endocytosis and upon endosomal acidification translocate their cargo LCs into the cytosol, where they gain access to their target substrates [26, 36].

A model for LC translocation has been proposed [26], whereby the lowering of pH in the endosome triggers a large conformational change in the translocation domain, resulting in formation of a channel in the vesicle membrane that then mediates translocation of the unfolded LC cargo domain through the channel into the cytosol, where it subsequently refolds. Whether and at what point further processing and dissociation of the receptor-binding domain (BD) from membrane-translocation domain (TD) occurs during the translocation process is not clear.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410985/


Nontoxic botulinum for drug delivery

Botulism is a severe and potentially fatal disease characterized by muscle paralysis. The causing agent, botulinum neurotoxins (BoNTs), has the ability to enter motor neurons and to block neurotransmission. In two independent studies, Miyashita et al. and McNutt et al. used nontoxic derivative of BoNT to deliver therapeutic antibodies against BoNTs in neurons. Miyashita et al. targeted BoNT/A and BoNT/B and reported therapeutic effects in mice. Using a similar approach targeting BoNT/A, McNutt et al. increased survival after lethal challenge in mice, guinea pigs, and monkeys. This approach provided a safe and effective treatment against BoNT intoxication and could be exploited for targeting other intracellular proteins in neurons.

Abstract

Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism.

Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin–based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol.

A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons.

Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice.

These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.

reference link : https://stm.sciencemag.org/content/13/575/eaaz4197


More information: Shin-Ichiro Miyashita et al, Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism, Science Translational Medicine (2021). DOI: 10.1126/scitranslmed.aaz4197

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