Re-engineering the target specificity of clostridial neurotoxins - a route to novel therapeutics

Foster, Keith A.; Adams, Emily J.; Durose, Lyndsey J.; Cruttwell, C J; Marks, Edwin P.; Shone, Clifford C.; Chaddock, John A.; Cox, Cassiah; Heaton, Chase M.; Sutton, J. Mark; Wayne, Jonathan; Alexander, Frances C.G.; Rogers, Duncan F.

doi:10.1007/bf03354881

Cited by 45 publications

(37 citation statements)

References 27 publications

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“…This proven technology should allow the creation of hybrid toxins optimized for specific interaction with different populations of neurons or other secretory cells; for example, exogenous ligands have been inserted into BoNT/A to replace H C (42,43).…”

Section: Discussionmentioning

confidence: 99%

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

Wang

Meng

Lawrence

et al. 2008

Journal of Biological Chemistry

106

131

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Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

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Section: Discussionmentioning

confidence: 99%

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

Wang

Meng

Lawrence

et al. 2008

Journal of Biological Chemistry

106

131

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“…Elucidation of the structure-function relationship of BoNTs has enabled the design of therapies that retarget BoNT to unique neurons and nonneuronal cells. Replacement of BoNT HCR domain with nerve growth factor, lectin from Erythrina cristagalli, or epidermal growth factors enable retargeting of BoNT/A to neuronal or nonneuronal cells such as nociceptive afferents and airway epithelium cells (13)(14)(15). However, the selective cleavage of neuronal-specific SNARE proteins by BoNT has limited development of therapies in these nonneuronal systems.…”

Section: Snap23 ͉ Snap25 ͉ Snare Proteinsmentioning

confidence: 99%

Engineering botulinum neurotoxin to extend therapeutic intervention

Chen

Barbieri

2009

Proc. Natl. Acad. Sci. U.S.A.

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Clostridium botulinum neurotoxins (BoNTs) are effective therapeutics for a variety of neurological disorders, such as strabismus, blepharospam, hemificial spasm, and cervical dystonia, because of the toxin's tropism for neurons and specific cleavage of neuronal soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNARE) proteins. Modifying BoNT to bind nonneuronal cells has been attempted to extend therapeutic applications. However, prerequisite to develop nonneuronal therapies requires the retargeting the catalytic activity of BoNTs to nonneuronal SNARE isoforms. Here, we reported the engineering of a BoNT derivative that cleaves SNAP23, a nonneuronal SNARE protein. SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of airway mucus, antibody, insulin, gastric acids, and ions. This mutated BoNT/E light chain LC/E(K 224 D) showed extended substrate specificity to cleave SNAP23, and the natural substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein delivery into cultured human epithelial cells, LC/E(K 224 D) cleaved endogenous SNAP23, which inhibited secretion of mucin and IL-8. These studies show the feasibility of genetically modifying LCs to target a nonneuronal SNARE protein that extends therapeutic potential for treatment of human hypersecretion diseases.Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans (1). BoNTs elicit neuronal-specific flaccid paralysis by targeting neurons and cleaving neuronspecific soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptors (SNARE) proteins. BoNTs are organized into 3 functional domains: an N-terminal zinc-metalloprotease light chain (LC), a translocation domain (HCT), and a C-terminal receptor binding domain (HCR) (1, 2). BoNTs bind luminal domains of synaptic vesicle proteins, upon the fusion of synaptic vesicles with the plasma membrane (3-5). BoNTs are internalized into endosomes and upon acidification, the LC is translocated into the cytoplasm, where SNARE proteins are cleaved (1, 2).Mammalian neuronal exocytosis is driven by the formation of protein complexes between the vesicle SNARE, VAMP2, and the plasma membrane SNAREs, SNAP25 and syntaxin 1a (6). There are 7 serotypes of BoNTs (termed A-G) that cleave specific residues on 1 of 3 SNARE proteins: serotypes B, D, F, and G cleave VAMP-2, serotypes A and E cleave SNAP25, and serotype C cleaves SNAP25 and syntaxin 1a (1). Thus, neuronal specificity is based upon BoNT binding to neurons and cleaving neuronal isoforms of the SNARE proteins. For example, BoNT/A cleaves human SNAP25, but not the human nonneuronal isoform SNAP23 (7,8). The nonneuronal SNARE isoforms are involved in divergent cellular processes, including fusion reactions in cell growth, membrane repair, cytokinesis, and synaptic transmission (reviewed in 9).The reversible nature of muscle function after BoNT intoxication that replace toxin-affected nerves with new nerves (10) has turned the BoNT from a deadly agent to therapies for neu...

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“…The resulting molecule should retain the attributes of both the LC catalytic function and the h N translocation function. This concept has currently been explored in relation to chronic pain and mucus hypersecretion, see the paper by Foster et al (2006). …”

mentioning

confidence: 99%

Botulinum neurotoxin — from laboratory to bedside

Foster¹,

Bigalke

Aoki

2006

neurotox res

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Botulinum neurotoxin (BoNT) has been used clinically since 1980, with an ever-increasing range of clinical applications. This has coincided with a period of massively expanded interest in the underlying biology of the neurotoxin. Tremendous advances have taken place in the scientific understanding of neurotoxin structure and function since the description of their endopeptidase activity in 1992. These developments have led to an increased understanding of the mechanisms underpinning the clinical use of the neurotoxins and also in the technologies available to support their clinical use. The expanding range of clinical applications, and use in increasing doses, has also generated challenges for the clinicians and manufacturers of BoNT preparations to ensure continuing efficacy and safety margins for these new clinical settings. To date the increased clinical use of BoNTs has occurred largely empirically, and not by application of the recent insights into neurotoxin structure and function. With the increased knowledge regarding the biology of the neurotoxins, however, there is the opportunity to select preferred forms of the toxin for particular clinical applications and even to consider engineering the neurotoxins to produce modified products more suited to specific clinical applications. These developments and opportunities that have arisen, particularly over the last decade, emphasise the increasing need to maintain an active two way dialogue between clinicians and basic scientists to ensure that the advances in the laboratory are translated into clinical benefit and that the clinical developments in use of neurotoxin are supported by the scientific research activity. This article is based upon presentations given in a workshop at the 5th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxin in Denver in June, 2005 seeking to address issues relating to the laboratory/clinic interface.

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Re-engineering the target specificity of clostridial neurotoxins - a route to novel therapeutics

Cited by 45 publications

References 27 publications

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

Engineering botulinum neurotoxin to extend therapeutic intervention

Botulinum neurotoxin — from laboratory to bedside

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