Clostridial neurotoxins, the botulinum neurotoxins (BoNT), of which there are seven serotypes (A to G), and tetanus toxin, are potent inhibitors of the vesicular secretion of neurotransmitters from nerve cells. The neurotoxins comprise three distinct protein domains, each of which plays a key role in the inhibition of vesicle fusion in target neurons (Figure 1 and Figure 2):
• A binding domain (HC), responsible for high affinity selective binding to the target neurons
• A membrane translocation domain (HN), that facilitates entry of the third domain into the cytosol
• An endopeptidase domain (LC), which cleaves the SNARE proteins thereby inhibiting neurotransmission (vesicular secretion)
The first two domains reside within a single 100 kDa polypeptide called the heavy chain (HC). The third, protease, domain resides in a separate 50 kDa polypeptide called the light chain (LC). The LC is linked to the HC via non-covalent interactions and a covalent disulphide bond. The LCs from different serotypes cleave different SNARE proteins, each at a unique peptide bond. Botulinum serotypes A, C and E all cleave SNAP-25, albeit at a different peptide bond. Tetanus toxin and botulinum serotypes B, D, F and G all cleave VAMP again at distinct peptide bonds. Botulinum neurotoxin serotype C is so far the only neurotoxin with a second substrate identified: syntaxin. Cleavage prevents formation of a productive SNARE complex and so inhibits of secretion.
In addition to cleaving different SNARE proteins, the different serotypes have different durations of action. Serotype A is the longest acting with a duration of action of 3 to 6 months in human motor-neurons. Serotype E has the shortest duration of action of 3 to 4 weeks in the same target. The duration of action of the neurotoxins is a property of the endopeptidase domain and the cleavage of the SNARE proteins, and so will be retained in Syntaxin’s TSI.
Delivered locally, the properties of selectivity, potency and prolonged activity, enables botulinum neurotoxin to have a profound therapeutic effect that is long in duration. Spread of the toxin or systemic exposure, however, has the potential to be seriously toxic through widespread muscular relaxation or paralysis, meaning that the native toxins have a very narrow therapeutic index. They are also only effective in specific neuronal cell types due to the restrictions of their natural targeting domain, thereby limiting their clinical utility to selected hypersecretory disorders of the peripheral nervous system. By contrast, Syntaxin’s TSI technology is not limited by this narrow therapeutic window nor is it restricted to this limited cellular population.
Syntaxin possesses in-depth understanding of the selectivity of the endopeptidases for specific SNARE proteins. By examining the SNARE protein composition of different cells types that are important in diseases, and linking particular SNARE proteins to the mechanism of secretion of a target hormone or other chemical mediator, Syntaxin can design and construct novel recombinant proteins (TSI) with the capacity to target cell secretion pathways quite specifically.
The ability to achieve cell specific targeting via a suitable targeting domain to a receptor or cell surface marker of restricted distribution reduces the potential for off target effect and ensures that delivery of endopeptidase and inhibition of secretion only occurs in the intended target cells. This means that, unlike the clinical botulinum neurotoxin products, Syntaxin’s TSI have the potential to be administered safely via systemic (e.g. i.v.) as well as by local routes of administration.
Figure 1: Illustration of the mechanism of action of BoNT in a nerve cell