Duplexed, bead-based competitive assay for inhibitors of protein kinases.
BACKGROUND
Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule.


METHODS
Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness.


RESULTS
A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening.


CONCLUSIONS
Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.
Simons
Peter C
PC
New Mexico Molecular Libraries Screening Center, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Young
Susan M
SM
Gibaja
Veronica
V
Lee
Wen-Cherng
WC
Josiah
Serene
S
Edwards
Bruce S
BS
Sklar
Larry A
LA
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001667:Binding, Competitive; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008863:Microspheres; D009532:Nickel / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
C471213:His-His-His-His-His-His; D008024:Ligands; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D009532:Nickel; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C498932:ACVR1B protein, human; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I