A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Abstract

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 hydrogelimmobilized 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 semi-quantitative detection of peptide phosphorylation by recombinant cAbl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay were 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.

Cell Treatment and Lysis in 96-Well Filter-Bottom Plates for Screening Bcr-Abl Activity and Inhibition in Whole-Cell Extracts

“…The Z factor considers both dynamic range and variability across multiple samples, with a value of 1 representing a theoretical perfect assay and values over 0.5 being suitable for high-throughput screens. In prior work using either purified c-Abl or Bcr-Abl in bulk K562 lysates, 8 we attained Z factors between 0.5 and 0.9 for the solid-phase kinase assay. In our present study, we were able to obtain a Z factor of 0.4, most likely due to well-to-well variations in the protein concentrations.…”

“…8 It provides a simple and robust platform to screen small molecules for inhibition of Bcr-Abl in vitro. To accelerate the examination of Bcr-Abl inhibition in cells, we developed a protocol to treat K562 cells in 96-well filter-bottom plates with various small-molecule inhibitors.…”

A Peptide-Based Biosensor Assay To Detect Intracellular Syk Kinase Activation and Inhibition

“…The captured peptide was then incubated with an anti-phosphotyrosine primary antibody (4G10) followed by a horseradish peroxidase-conjugated secondary antibody. Chemifluorescent detection was accomplished by incubating each well with Amplex Red™ reagent and hydrogen peroxide, which gives a fluorescence signal readout that is proportional to the amount of horseradish peroxidase-conjugated antibody in each well, and thus reports the degree of phosphotyrosine present (via the anti-phosphotyrosine antibody) (as described by Wu et al 19 ). Relative fluorescence units (RFU) were measured, which provided a proportional representation of the amount of phosphorylated biosensor present.…”

124I-Iodopyridopyrimidinone for PET of Abl Kinase–Expressing Tumors In Vivo

“…Recently, a solid-phase PKI screening method in multiwell plates was suggested using an imatinib analog (42). Displacement studies reported in the article demonstrate one of the potential routes to the in vitro application of the radiolabeled SKI230.…”