An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases.
We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.
Fowler
Ann
A
Lead Generation Biology, AstraZeneca R & D Charnwood, Loughborough, UK.
Swift
Denise
D
Longman
Emma
E
Acornley
Anne
A
Hemsley
Paul
P
Murray
Dave
D
Unitt
John
J
Dale
Ian
I
Sullivan
Elaine
E
Coldwell
Martin
M
D051698:Activating Transcription Factor 2; D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein / Q000737:chemistry; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D048031:JNK Mitogen-Activated Protein Kinases; D008851:Micropore Filters; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D014157:Transcription Factors / Q000032:analysis / Q000037:antagonists & inhibitors
D051698:Activating Transcription Factor 2; D000911:Antibodies, Monoclonal; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019793:Fluorescein