Silencing genes at either the DNA or RNA level is among the most popular tricks in the cell biologist's toolbox. And in the Sept. 8, 2006, issue of Cell, researchers from Stanford University completed the set, reporting on, as the title of their paper states, "A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules."

Senior author Thomas Wandless listed several advantages that the new system has over silencing at the DNA or RNA level. For one thing, the degron system is an order of magnitude faster: It works in two to four hours, while with DNA or RNA interference it typically takes 24 to 36 hours until the protein is truly gone from the cell.

"Another thing about [DNA and RNA targeting methods] is that they're really not tunable," Wandless told BioWorld Today. "Theoretically they could be, but in practice they are binary."

To develop a tunable system that works at the protein level, the scientists used a mutated FKBP 12 protein. "Wild-type FKBP is perfectly stable," Wandless said. But change a few amino acids in its sequence, and it turns into a "degron" - a phrase first coined in 1994 to describe a similar system to degrade proteins. Wandless said that for his team's more complex system, "ligand-dependent conditional degron is a good descriptor." Not only is the degron rapidly degraded itself, but if you fuse it to another protein, that other protein will be chewed up, as well.

The scientists chose FKBP as a candidate destabilizing domain because it already is a well-studied protein with its share of ligands, and because neither the protein nor the ligands will wreak havoc on cells they are introduced to. The trick to finding the best combination was to not go overboard.

FKBP is "very easy to mess up a lot," said Wandless, who is an assistant professor of molecular pharmacology at Stanford. "But we needed to find a sweet spot where it's recognized as abnormally folded in the absence of our drug, but stable in the presence of our drug." By screening a library of FKBP mutations, testing them with a group of known ligands and their derivatives, his group identified combinations that met this double requirement.

Wandless named several applications that the degron system might enable. In the short term, direct clinical applications are unlikely; Wandless said that "down the road, when the world gets a little more comfortable with gene therapy," it could theoretically be used in applications there to provide patients with proteins that can be turned off. "But that requires so many things, in humans, that it is not even really worth thinking about at this point," he added.

In the meantime, though, there are several basic science applications to keep researchers entertained. Wandless said that the approach harnesses the benefits of small molecules without the need for screening: "Small molecules are very powerful tools, provided Mother Nature has provided you with one that is specific," he said. And the degron system makes Shield-1 specific to whichever protein a scientist wants: "All they have to do is know how to make fusion proteins."

The system also could be useful in target validation. "Fundamentally, you now have a way to knock down or inhibit a protein in a dose-dependent way," Wandless said. "The small-molecule control gives you the same dose dependence that you see with anything in your medicine cabinet," and that makes it unique.

On the development side, Wandless and his team are working on making a similar system that works in yeast; they also are looking to extend the system by finding a second small molecule-protein combination that works like FKBP12 and Shield, which would open up the possibility of doing a range of protein interaction studies with the system.

Researchers, though, already seem to have plenty of ideas for what to do with just one mammalian cell FKBP: Wandless said that he has been getting about five requests for the system a day.