FRET-based drug screen detects kinases

David Shenkenberg

Kinases switch cellular pathways on and off by phosphorylating cellular components. They are ideal drug targets because drugs that target them can control entire pathways. Although Förster resonance energy transfer (FRET) can easily monitor kinase activity in single cells, it cannot easily measure entire compound libraries. Researchers have solved that problem by developing a FRET-based high-throughput drug screen that detects the activity of protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP), the latter of which activates PKA.

Scientists in Jin Zhang’s laboratory at Johns Hopkins University School of Medicine in Baltimore engineered new FRET-based reporters by integrating modified versions of yellow and cyan fluorescent proteins with proteins that interact with PKA and cAMP.

Zhang said that they chose FRET because it is noninvasive, sensitive and provides spatiotemporal resolution. She added that they could genetically encode fluorescent proteins in the cells used in their screen, which they could not do easily with fluorescent dyes. However, dyes tend to emit light more intensely than fluorescent proteins and have a greater dynamic range, so the researchers circularly mutated the Venus domains of the proteins to make them brighter and to increase their signal amplitude.

This FRET-based drug screen monitors kinase activity. Each well of the plate contains living mammalian cells expressing a kinase biosensor. A plate reader measures cyan and yellow fluorescence, and a computer calculates the emission ratios, resulting in a graph showing responses generated by individual compounds.

When performing FRET assays with the reporters, the fluorescence measurements lasted 64 to 92 seconds. Zhang said that her group is attempting to hasten the process by devising a method to simultaneously detect both fluorophores.

The researchers next modified the PKA indicator with a nuclear export signal because kinase activities often are compartmentalized. They saw an expected difference in kinetics due to the exclusively cytosolic location of the reporter. Zhang said that they have developed a version of the reporter with a nuclear localization signal, and they are currently testing it.

They found that the PKA indicator exhibited high sensitivity and low variability, but that the cAMP reporter initially showed only acceptable levels of sensitivity and variability. However, they improved results for the latter indicator by stably transfecting the cells with the construct.

They validated their assay on a 160-drug library of FDA-approved drugs and clinically relevant compounds, including lidocaine, coumarin, aspirin and testosterone. All known agonists and antagonists produced positive hits, and most drugs showed no effect. The researchers said that these results prove the accuracy of their screen.

However, a few drugs confounded the results because of their toxicity or colorimetric properties. In addition, bilirubin unexpectedly inhibited cAMP. Although the researchers added a high concentration of bilirubin, they suggested that their results could indicate a new mechanism of bilirubin activity.

Before looking at interesting hits individually, the researchers plan on continuing to test the rest of the compounds in the library, which number more than 3000.

ACS Chemical Biology, July 26, 2006, pp. 371-376.