Most people know botulism as a terrible disease and botox as a wrinkle reducer that can give people a 'frozen' appearance if it's used to excess. But these things are all made possible by the same 'miracle' toxin: botulinum, which is produced by the bacterium Clostridium botulinum and related species. The toxin has medicinal applications if it's used in the right dose. It doesn't only restore a youthful appearance, it can also treat chronic migraines, some chronic muscle spasms, and uncontrolled blinking.
Reporting in Science, researchers have now reported a way to modify the botulinum toxin to open up more therapeutic applications. They were able to engineer the toxin protein to create unique, highly selective proteases, which are enzymes that can make cuts in other proteins that may activate or degrade those proteins. These modified proteases could be useful in the treatment of inflammation, aid in the growth of new neurons, or control growth hormone levels, for example, with many other potential applications.
"In theory, there is a really high ceiling for the number and type of conditions where you could intervene," said first study author Travis Blum, a postdoctoral researcher in the Department of Chemistry and Chemical Biology.
The research team was able to reprogram proteases so they could cut different targets in specific proteins, even when these proteins bore little or no similarity to the original protease target, and without cross-reactivity. They also began to find ways to create therapeutics that could be sent to the inside of a cell, what Blum called a "classical challenge in biology." Large amounts of botulinum toxin can move into neurons, making them even more useful.
It's now possible to make unique proteases directed at specific proteins. "Such a capability could make editing the proteome feasible in ways that complement the recent development of technologies to edit the genome," suggested David Liu, the Thomas Dudley Cabot Professor of the Natural Sciences, a Howard Hughes Medical Institute Investigator, and a core faculty member of the Broad Institute.
Proteins or proteases could act as therapeutics in various ways, such as modifying other proteins to trigger a downstream effect, or to eliminate other destructive or defective proteins, for example.
"Despite these important features, proteases have not been widely adopted as human therapeutics," said Liu, "primarily because of the lack of a technology to generate proteases that cleave protein targets of our choosing."
Liu's lab developed PACE (phage-assisted continuous evolution), which generates novel proteins without needing much maintenance from people. PACE is a system in which the evolution of a desired protein can be driven or directed by the continuous infection of a bacteria over generations by a bacteriophage, which induces changes in the bacterial genome (and hence, the proteins it makes). The scientists were able to use PACE to make so-called 'promiscuous' proteases more selective about which proteins they cut. Next, they engineered a protease that would identify an entirely new target.
"At the outset, we didn't know if it was even feasible to take this unique class of proteases and evolve them or teach them to cleave something new because that had never been done before," said Blum. But it worked.
They've made four proteases from three botulinum toxin families; all four stopped acting on their original targets and began specifically cutting new targets, all while maintaining the ability to get into cells. "You end up with a powerful tool to do intracellular therapy," said Blum.
Blum cautions that this work is still theoretical, because these proteases are not ready for use in the clinic; far from it. These new proteases might stimulate an immune response, for one thing. The team is now working to learn more about whether they can use other proteins that are less likely to trigger an immune attack as their starting material. They are also continuing to improve the botulinum toxin proteases.
"We're still trying to understand the system's limitations, but in an ideal world, we can think about using these toxins to theoretically cleave any protein of interest," said Blum.
Sources: AAAS/Eurekalert! via Harvard University, Science
