High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.
BACKGROUND
High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.


RESULTS
Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity.


CONCLUSIONS
The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.
Rininsland
Frauke
F
QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507, USA. frauke@qtlbio.com
Stankewicz
Casey
C
Weatherford
Wendy
W
McBranch
Duncan
D
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis; D002417:Cattle; D018797:Cell Cycle Proteins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006657:Histones / Q000032:analysis; D006801:Humans; D020128:Inhibitory Concentration 50; D053592:Interleukin-1 Receptor-Associated Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D007477:Ions; D004676:Myelin Basic Protein / Q000032:analysis; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods; D051571:Protein Kinase C-alpha / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D011506:Proteins / Q000737:chemistry*; D040641:Quantitative Trait Loci; D020539:Sequence Analysis, Protein / Q000379:methods*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D005456:Fluorescent Dyes; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D007477:Ions; D004676:Myelin Basic Protein; D019151:Peptide Library; D010455:Peptides; D010750:Phosphoproteins; D011108:Polymers; D011506:Proteins; D010770:Phosphotransferases; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D017346:Protein Serine-Threonine Kinases; D051571:Protein Kinase C-alpha