Engineering botulinum neurotoxin to extend therapeutic intervention

Abstract

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...

Proteases as therapeutics

“…Botulinum toxin type A is in clinical trials for numerous additional indications, including pain, muscle spasticity, overactive bladder, alopecia areata, benign prostatic hyperplasia and headaches, whereas type B is in clinical trials for spasticity due to cerebral palsy and cervical dystonia [93]. Besides the development of botulinum toxin for more indications, other efforts are aiming to reduce immunogenicity of the material, developing less invasive delivery methods and improving stability of the drug [94,95]. …”

Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery

“…In fact, Botox™ (BoNT/A) treatment is FDA approved, and according to statistics provided by the American Society for Aesthetic Plastic Surgery [11], is one of the top non-surgical cosmetic procedures performed in the United States. Furthermore, to extend BoNT potency for clinical neurology applications, chimeric and modified BoNTs have been generated using protein engineering methods to expedite the toxin’s cellular entry, increase its substrate specificity, and extend its duration of activity in neuronal and possibly non-neuronal cells [9, 12–14]. …”

Re-Engineering Clostridial Neurotoxins for the Treatment of Chronic Pain

“…Although BoNT-A has been considered the obvious serotype to form the basis of engineered proteins, studies of the mechanism by which the light chain of BoNT-A recognizes and cleaves SNAP25 have shown that the light chain of BoNT-A requires a longer substrate and a greater number of residue interactions than, for example, BoNT-E. [95] As BoNT-E has a more straightforward interaction with SNAP25, this may make a more suitable toxin to engineer, in particular, for studies seeking to alter the substrate specificity and extend the therapeutic application beyond neurologic conditions. [96] However, for reasons that have yet to be established, the duration of action of BoNT-E is much shorter than that of BoNT-A, which is an additional important consideration when identifying the most suitable molecule to modify for clinical application.…”