InteliSpark client, Lucidicor Inc. has been awarded a Phase I SBIR from the National Institute of General Medical Sciences for their project, “Novel Fluorescent Biosensors to Continuously Visualize Real-Time Protein Phosphorylation in Live Cells”. This project will focus on developing technology that would enable detection of phosphorylation continuously in real time within living cells and able to adapt to multiple kinases.
Protein phosphorylation is essential in orchestrating the myriad aspects of cell function. Kinases, which phosphorylate proteins, are encoded by 2% of the human genome and are critically involved in diverse conditions such as cancer, metabolic disorders and neurodegeneration. Current tools for detecting cellular phosphorylation are limited because most tools examine this process at a single time point or in cell lysates, or are restricted to single kinases. Lucidicor Inc. has developed a technology, PhosFluorTM, an extensively engineered protein that fluoresces robustly only when phosphorylated. Currently, this is the only fluorescent protein to date with this property. By engineering substrate recognition sites into PhosFluor, this molecule can be converted into a biosensor for the activity of virtually any protein kinase. Using PhosFluor, Lucidicor has already created and validated biosensors for Protein Kinase A (PKA), cyclin- dependent kinases (cdks), and Src-family kinases. A 700% change in the fluorescence of PhoFluor biosensors is observed when cells are stimulated physiologically. By contrast, dual-color fluorescent protein biosensors that utilize FRET (Förster Resonance Energy Transfer) exhibit a small 25-30% change in overall fluorescence during a physiological stimulus. The higher responsiveness of PhosFluor greatly optimizes fluorescence detection and signal/noise, which are essential for consistently accurate measurements within cells.
Lucidicor Inc. envisions that the utility of their PhosFluor will be greatly enhanced if it is integrated with other methods. Specifically, integration of PhosFluor with advanced microscopy would allow precise visualization and measurement of the spatio-temporal action of kinases. Integration with viral transduction would permit analyses of kinase activity in diverse cell types, which in turn has potential in diagnostics or drug discovery. In Phase II, they plan to extend the utility of our biosensors to neurons, primary cells, and human specimens. PhosFluor's low background and high signal/noise lends itself to high throughput applications, such as the capture of information in microscopy-based high content screening. Collectively, this will enable creation of a kinome toolbox that can be used by basic and clinical investigators, and the pharmaceutical industry.