Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy.
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 microl and on the single microl 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' = approximately 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-microl final volume against company file compounds.
Wright
Penny A
PA
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.
Boyd
Helen F
HF
Bethell
Richard C
RC
Busch
Michael
M
Gribbon
Phillip
P
Kraemer
Joachim
J
Lopez-Calle
Eloisa
E
Mander
Thomas H
TH
Winkler
Dirk
D
Benson
Neil
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000918:Antibody Specificity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D016014:Linear Models; D048688:MAP Kinase Kinase 7; D008274:Magnesium / Q000378:metabolism; D008832:Microchemistry / Q000379:methods*; D048057:Mitogen-Activated Protein Kinase 10; D048055:Mitogen-Activated Protein Kinase 8; D048056:Mitogen-Activated Protein Kinase 9; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry; D012680:Sensitivity and Specificity
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D004791:Enzyme Inhibitors; D008024:Ligands; D010446:Peptide Fragments; D012235:Rhodamines; D000255:Adenosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10; D048056:Mitogen-Activated Protein Kinase 9; D011505:Protein-Tyrosine Kinases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases; D008274:Magnesium