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DOI: 10.1177/108705702237673 Development of a 1-µl Scale Assay for Mitogen-Activated Kinase Kinase 7 Using 2-D Fluorescence Intensity Distribution Analysis AnisotropyPfizer Global Research and Development, Sandwich, Kent, United Kingdom.
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.
Evotec OAI, Hamburg, Germany
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.
Evotec OAI, Hamburg, Germany
Evotec OAI, Hamburg, Germany
Evotec OAI, Hamburg, Germany
Evotec OAI, Hamburg, Germany
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom. This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 µl and on the single µl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = ~0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-µl final volume against company file compounds.
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