Development of a Fluorescence Polarization AKT Serine/Threonine Kinase Assay Using an Immobilized Metal Ion Affinity-Based Technology

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Abstract
A bead-based FP assay methodology, termed IMAP trade mark, has been developed for the serine/threonine kinase, AKT, that allows for direct measurement of product formation. The assay design utilizes a fluoresceinated peptide substrate that, when phosphorylated by the kinase, binds to nanoparticles derivatized with trivalent metal cations through a metal-phospholigand interaction. The result of this bound fluoresceinated phosphorylated product is an increase in polarization signal caused by a decrease in the molecular mobility of the bound product. The AKT IMAP FP assay has been formatted in a 384-well microtiter plate with a Z' of 0.75, suitable for HTS. The assay was validated with six known kinase inhibitors. The IC(50) values generated were comparable to previously reported values using a competitive antibody-based FP assay and a radioactive [(33)P]ATP Flashplate transfer assay. The IMAP assay was further evaluated by screening the biologically active sample set, LOPAC trade mark. It was found that no fluorescent samples interfered with the assay's performance and that one could identify appropriate inhibitors. The advantages of IMAP technology are that it does not require the use of antibodies, the polarization signal generated is large in comparison with most FP assays based on antibodies, and IMAP captures and measures the product formed directly. The higher concentrations of fluorophore used in the assay versus competition assays increase the precision of the data obtained and reduce sample interference from compounds. This work demonstrates that IMAP is a valuable technology that may be used in developing numerous kinase assays.