Toward the development of real-time cellular kinetic assays to characterize inhibitors of cytokine signalling
A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The realtime kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in 2 vitro, and because cytokines receptors differentially utilize their
... y utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-γ) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-γ receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-γ signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.