One of the deadliest weapons in the biowarfare arsenal is on its way to becoming a billion-dollar medicament and cosmetic.

During World War II, the U.S. military manufactured botulinus-toxin bombs (as well as anthrax), expecting Nazi Germany to do the same. A soil bacterium, Clostridium botulinum, manufactures the neurotoxin full-time. Nowadays, it's best known in food-poisoning (especially home-canning products), infant infection and wound injury.

Once lodged inside a neuron, the toxin brings on paralysis and frequently swift death.

"What you're seeing in biotechnology," observed structural biologist and biomaterials scientist Raymond Stevens, "is that protein molecules like this are being used more and more as therapeutics. Botulinus toxins are like a pair of very specific molecular scissors; they have powerful applications. But one needs to be very careful, and understand how these molecules work, as their use continues to grow."

Because it acts on muscle function, botulinus toxin finds medical applications in treating involuntary muscle disorders such as dystonia and Parkinson's disease.

"Sales of botulinum neurotoxin are expected to exceed a billion dollars in the coming years," Stevens observed, "as a therapeutic and cosmetic — which is growing extremely fast."

He explained: "Stanford University has a program where they'll inject the toxin into muscles. This reduces muscle tension and relieves wrinkles. It's a crazy idea," Stevens added, "but it seems to work very well, and it's one of the things that seems to be driving up sales."

Stevens, who is in the chemistry department at the University of California's Berkeley National Laboratory, is senior author of a paper in the current issue of Nature Structural Biology, dated Oct. 2. Its title is "Crystal structure of botulinum neurotoxin type A and implications for toxicity."

He and his co-authors have been working on this project full-time for the past six years, with funding from the U.S. Army Medical Research Institute of Infectious Diseases, at Fort Detrick, Md.

Stevens described the unique reason it took so long:

"One of the problems we had was that we can't genetically engineer the protein, or modify it, because it's a biological weapon. So that made things extremely difficult, given its toxicity.

"Ordinarily," he went on, "people like to clone and express things in something like E. coli — make mutations and so forth. We weren't able to do that, and we had a tremendous amount of difficulty working with the purified protein, because as a purified neurotoxin it's unstable.

"We had to work with it only from the original Clostridial organism," he recalled, "so we spent a lot of time trying to figure out alternative ways of purifying it to get high-resolution data, making heavy-atom derivatives, and so forth."

Strange, Lethal Modus Operandi Revealed

The team described a molecule differing widely in function as well as form from other known toxins.

"What the bacterium has done," Stevens related, "is develop an absolutely beautiful mechanism for making sure that it shuts down neuromuscular signaling. Its toxic complex is able to go completely through our bodies, and survive all of our defenses, then deliver its toxin to neural cells. Inside those cells, different antigenic serotypes go after different synaptic vesicle proteins.

"It makes sure that those proteins are destroyed, so that they can't function in intramuscular signaling. And that's what causes paralysis. It's interesting," he added, "because botulinum neurotoxins are extremely specific in targeting various synaptic vesicle proteins. As proteases, you'd expect them to have some degree of specificity for other molecules as well, but they don't. "

The co-authors' three-dimensional analysis of the protein's structure revealed much of its strange but lethal modus operandi.

"The structure is a hybrid of three different structural motifs," Stevens said, "which match three different mechanisms that the toxin uses for its travels. The first structural motif is that of a sugar-binding protein that's been observed for many other proteins. It's this binding domain that recognizes the neuronal cells, and adheres to their surfaces.

"The second motif," he went on, "is the translocation domain. This is not like any other toxin that we've seen before; it looks more like influenza virus, with long helices. It's this domain, upon a change in pH, that forms pores inside the membrane, once it gets inside of the cells. After it forms this pore, it is then able to release the third and last motif, which is the catalytic domain, into the cytoplasm.

"That catalytic domain is allowed to pass through the membrane to get inside of the cell, where it then acts as a protease, and this cleaves the synaptic vesicle proteins."

One feature in this novel pathway "calls for radically different inhibitor design strategies from those currently used," the Science paper states.

Stevens explained: "A lot of people would like to have inhibitors for this botulinum toxin, because it's used as a biological weapon, as well as a therapeutic. You'd like to be able to control its functions. So, there's a tremendous amount of effort going into trying to design inhibitors of the toxin's activity."

Inhibitors Needed That Hit Before Cell Entry

He pointed out the need for a new approach.

"The toxin's activity is as a protease, so most people have followed the standard protease-inhibitor design strategies. A lot of these are peptides, which look like proteins, but they put in a backbone that the protease cannot cleave — [a backbone] that it'll bind and recognize.

"Now the problem is that the active site for botulinum toxin, based on the structure, is hidden away. You can't gain access to it until the toxin actually gets inside of a cell and it's released from the rest of the protein. Sort of like a Trojan horse — you don't know what's inside until it's too late

"So, what you need to do is have inhibitors that are very small, that can actually get into this active site, before it actually gets inside the cell. Because once it gets inside, it's going to cause damage."

Stevens and his co-authors are working on small inhibitors such as those. "We're doing general design right now," he allowed. "But there seem to be quite few companies that are interested." *

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