Print ArticleScientists have used a glowing protein from fireflies to observe the activity of a molecule that is an important target for new drugs to treat cancer, autoimmune diseases and several other disorders.
The target molecule, known as IKK (for IKappa kinase), regulates processes that can trigger dramatic changes in cellular physiology. Scientists have linked these changes to many different disorders.
"Our new system allows researchers to monitor whether drugs for these conditions are hitting this exact molecular target in cell culture and laboratory animals," says senior investigator David Piwnica-Worms, M.D., Ph.D., professor of molecular biology and pharmacology and of radiology.
Piwnica-Worms and lead author Shimon Gross, Ph.D., a postdoctoral fellow, measured light from the firefly protein, luciferase, to monitor IKK activity in tumor cells and inflamed liver cells in live mice. They also showed that the technique can greatly reduce the costs of tests that establish the best dosages for drugs that target IKK. Their results appear in the August 2005 issue of Nature Methods.
IKK stands at a pivot point in the middle of an important set of linked chain reactions known as the NF-KappaB pathway. The pathway can start at many different receptors on cell surfaces; its finish changes the activity levels of varying genes. The result, according to Piwnica-Worms, is that the potential reaction patterns in the NF-KappaB pathway form an hourglass-like shape, fanning out among many options at the start, narrowing in the middle, and again fanning out among many options at the end.
"At the waist of that hourglass is IKK," he explains. "This appears to put it in a position to be the key regulator of the pathway, and that has made it a subject of great interest both from the perspective of understanding how this pathway works and from that of developing new drugs for conditions that involve this pathway. "
Piwnica-Worms' laboratory has previously developed techniques that use luciferase to monitor protein-protein interactions. Researchers can employ an instrument known as an in-vivo bioluminescence camera to take real-time measurements of light from luciferase in cell cultures and in cells within live animals.
To use the firefly protein to monitor IKK, Gross altered cell lines to genetically fuse the luciferase protein to IKB (IKappaB), the protein that comes immediately after IKK in the NF-KappaB pathway. When the pathway is enabled, IKK triggers reactions that lead to the degradation of IKB. In cells with genetically altered IKB, the attached luciferase is broken down too, meaning scientists can detect increased IKK activity via decreased light from the cells.
"This is like doing in-vivo pharmacodynamics and pharmacokinetics," says Piwnica-Worms in reference to the sciences that study the effects, distribution and dissipation of drugs. "Traditionally the only ways we could do those kinds of studies were either to test for levels of the drug in the blood or to label the drug with a radioactive tracer.
"In the case of NF-KappaB, there were also methods that monitored IKK activity via changes in the levels of gene activation at the end of the pathway," he notes. "But those took hours to days to deliver results, and our approach works continuously and in real time." In their study, Gross and Piwnica-Worms tested the technique in live mice by transplanting genetically altered tumor cells and by using a technique that inserted the fused IKB/luciferase protein into liver cells only. They are currently working to develop a line of mice with the IKB/luciferase fusion built into its genetic code.
In addition, they showed that the system is not only helpful for learning if a drug is having the desired effect, it can also be used to fine-tune drug dosage for maximum benefit.
"One of the reviewers of our paper suggested that we should use the system to produce a full dose-response curve, which helps establish how to best use a drug," Piwnica-Worms says. "Establishing that normally takes 6 months and 300 mice. With our monitoring technique, Shimon did it in a 5-day period using 30 mice. That's going to lead to tremendous cost savings."
Because the luciferase-based monitoring system allows monitoring in live animals, Gross could perform multiple tests on the same mouse over time. He was also able to monitor the mice for individual variances that could inappropriately bias the results.
Gross S, Piwnica-Worms D. Real-time imaging of ligand-induced IKK activation in intact cells and in living mice. Nature Methods, August 2005. Funding from the National Institutes of Health.
Source : Washington University School of Medicine
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