Wider therapeutic possibilities for botulinum toxin

Change to a single amino acid in botulinum toxin A (the active ingredient in Botox^?) causes it to target non-neuronal signalling proteins, potentially widening the product’s therapeutic applications, say US scientists.

Joe Barbieri and his group at the Medical College of Wisconsin, Milwaukee, modified part of the toxin - the ’light chain’ protease enzyme - which is responsible for cleaving a signalling protein called SNAP25. Cleavage of SNAP25 blocks the release of neurotransmitters from affected nerve cells, which causes paralysis. In therapeutic and cosmetic applications, this localised paralysis is used to suppress involuntary muscle contraction, control excessive sweating and decrease the appearance of wrinkles.

Expanding scope

Barbieri explains that SNAP25 is part of a larger family of proteins, called SNARE proteins, which are involved in a variety of cell signalling activities. He reasoned that if the protein-cleaving light chain of botulinum toxin could be modified to cleave the non-neuronal SNARE protein, SNAP23, then this could lead to effective therapies for a range of diseases based around oversecretion, such as asthma and chronic obstructive pulmonary disease (COPD), where excessive mucus builds up in the lungs.

To do this, the group had to work out why the natural toxin cleaves SNAP25 but doesn’t cleave SNAP23. ’It’s always been a puzzle as to why the toxin doesn’t cleave both,’ says Barbieri. ’We modelled the binding in the active site of the enzyme, and we noticed there was a charge repulsion when SNAP23 was inside. There were two basic residues, one on SNAP23 and one on the light chain, which could repel each other,’ he explains.

The team then modelled a modified version of the toxin’s light chain, in which a basic lysine in the active site was replaced with an aspartic acid residue, in the hope that changing this repulsion into an attraction would make SNAP23 a viable substrate for the toxin to cleave. Sure enough, when the team produced a mutant version of the light chain with this single amino acid changed, it could cleave both SNAP25 and SNAP23. Since SNAP25 is only present in neurons and SNAP23 in non-neuronal cells, the loss of specificity is not a problem as long as the enzyme can be targeted specifically.

To make this modified light chain into a useful treatment, the team now need to look at ways of targeting specific cells rather than just neurons, and are currently working on a delivery system: ’We’re trying to fuse [the modified light chain] to a receptor component [such as an antibody], which will target it to a set of cells, to see if it can specifically inhibit secretion of various proteins or signalling molecules from non-neuronal cells,’ says Barbieri.

With combined therapeutic and cosmetic sales of Allergan’s Botox^? treatments reaching $1.3 billion (?792 million) in 2008, the market for modified botulinum toxin treatments is potentially huge, particuarly if applications could be expanded into more mainstream disease areas such as asthma and COPD or inflammatory and immune disorders. However, Barbieri recognises that it will be a while before such treatments make it into the clinic. ’It all depends on how stable the light chain is when we fuse it to another protein, and that turns out to be pretty hard to predict,’ he explains.

Phillip Broadwith