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UNC Scientists Develop Allosteric Activation Technique for Targeted Control of Protein Kinases

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By Adam Bonislawski

Scientists at the University of North Carolina have developed an allosteric activation technique for controlling the activity of protein kinases in living cells that they claim offers advantages over other methods for studying kinases, such as small-molecule inhibitors or genetic knockouts.

The technique, which the team detailed in a paper published online last week in Nature Biotechnology, allows researchers to turn specific kinases on and off, enabling expanded study of these key regulatory proteins, Klaus Hahn, professor of pharmacology at UNC and one of the paper's lead authors, told ProteoMonitor.

Kinases are central to a number of cellular processes and have been implicated in a wide variety of diseases, including cancer. In a recent interview with ProteoMonitor, Steven Pelech, president of bioinformatics firm Kinexus, estimated that some 80 kinases are currently considered drug targets and roughly one-third of drugs in preclinical and clinical trials target kinases.

"If you were to pick a few central regulators of all cell physiology, kinases would be one of them," Hahn said. "They have their fingers in everything. So it's a good one to go after."

The method Hahn's group developed to control the activity of these proteins is called engineered allosteric activation. It works through the insertion of the rapamycin-binding peptide insertable FKBP – iFKBP – into the kinase of interest. DNA coding for iFKBP is inserted into a clone expressing the target kinase. The insertion of this peptide increases the flexibility of the kinase's catalytic domain, resulting in a loss of activity. To restore the kinase's activity rapamycin is added, which binds to the inserted iFKBP peptide, restoring rigidity to the kinase's catalytic domain.

The technique has several advantages over conventional methods for studying kinases, Hahn said.

"If you want to study kinases now, you use inhibitors, which are not that specific," he said. "So if you have extremely related kinases, like, for example, the SRC family kinases, which are very important in cancer and very closely related but each with a different role, you can't really do that with inhibitors."

Engineered allosteric activation, on the other hand, allows researchers to alter a specific single kinase so that it can be controlled via the addition of rapamycin.

It also allows researchers to time precisely when in a cellular process they want to activate a kinase they are studying, an advantage, Hahn said, over current genetic approaches to kinase study.

"There are genetic [approaches] where you knock out the kinase or you replace it with mutants and you ask about specific aspects of the function. But you would often have compensation. You wouldn't be able to study the cells or animals until 24 hours later or so, so they would up-regulate other molecules to compensate."

The allosteric approach, on the other hand, gives some degree of control over the process. "You can throw [rapamycin] into the medium and control with pretty precise timing when the cell is affected. So if you're doing immune surveillance or looking at different stages of metastasis you can ask when your kinase is involved, in which stage, unlike genetic approaches where you just turn the whole thing off," he said. "It combines the control of timing and convenience of an inhibitor with the absolute specificity of genetics."

Because the iFKBP peptide is inserted into a portion of the target kinase that is highly conserved, Hahn expects that the technique will work across the entire class of proteins. So far his team has used it to control seven different types of kinases, he said. Presently he is using it to study SRC and MAP kinases as part of his lab's work on metastasis.

Discovery of the technique was something of a happy accident, Hahn said. The researchers had been trying to inhibit kinase activity by binding rapamycin to an iFKBP peptide inserted near the protein's active site. When as a control they inserted iFKBP in the kinase at a spot far removed from its active site they found to their surprise that the opposite happened — the insertion knocked out activity, which the addition of rapamycin then restored.

The researchers have patented the technique and have received some interest from undisclosed commercial firms regarding licensing, Hahn said, noting that the method's simplicity makes it a good candidate for commercialization.

"We have a history in my lab of developing techniques, some of which have been commercialized, and this appears to be by far the easiest," he said. "It's extremely robust."

He noted that with other technologies the lab has developed, "we were almost like technical [support] people on the phone trying to get them to work, and we realized that those techniques would never be that commercializable for that reason. But this thing so far just works. Put it in the kinase. Put in the rapamycin. It's really simple."

The technique could be particularly useful in drug development, Kahn said, particularly given pharma's interest in kinases.

"I think it has a lot of applications in high-throughput screening," he said. "If you wanted to look for drugs that have an effect downstream of a given kinase you could easily have 96-well plate assays with living cells and your robot could just shoot rapamycin into each well to activate the kinase and you could look at the effects on a downstream readout."

He's also received interest in the technology from a number of academic labs, Kahn said.

"After the paper came out we got a lot of requests for other kinases, so I'm hoping that other people will use it to explore other areas of biology that we're not exploring. The point about this method is that it's general," he said.

The team hopes to generalize the method even further by modifying it for use on other classes of proteins.

We're exploring additional applications and modifications of it to extend the capabilities to other proteins. It's really early stages, but that's what we're trying to do now," he said.

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