A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates.
Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.
Wu
Ding
D
Ludwig Center for Metastasis Research, University of Chicago, Chicago, IL 60637, USA.
Mand
Michael R
MR
Veach
Darren R
DR
Parker
Laurie L
LL
Clarkson
Bayard
B
Kron
Stephen J
SJ
D001681:Biological Assay / Q000379:methods*; D002457:Cell Extracts; D004353:Drug Evaluation, Preclinical; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000378:metabolism*; D020014:K562 Cells; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013997:Time Factors
D002457:Cell Extracts; D010455:Peptides; D047428:Protein Kinase Inhibitors; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D016044:Fusion Proteins, bcr-abl