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

Analytical Biochemistry
Ding WuStephen J Kron

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 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 spe...Continue Reading

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Citations

Feb 9, 2012·The Analyst·Angela ProctorNancy L Allbritton
Jul 10, 2012·Molecular BioSystems·Guangchang ZhouStephen J Kron
Apr 29, 2010·Molecular Cancer Therapeutics·Juliesta E Sylvester, Stephen J Kron
Sep 3, 2010·Analytical Biochemistry·Guangchang ZhouStephen J Kron
Jan 25, 2017·Proceedings of the National Academy of Sciences of the United States of America·William J NetzerVictor Bustos
Oct 20, 2012·Chembiochem : a European Journal of Chemical Biology·Yang DengBenjamin E Turk
Jul 2, 2010·Biopolymers·Ding WuStephen J Kron
Mar 25, 2010·Cancer Research·Kaushal Parikh, Maikel P Peppelenbosch
Jan 6, 2010·Journal of Nuclear Medicine : Official Publication, Society of Nuclear Medicine·Mikhail DoubrovinSteven M Larson
Sep 24, 2010·Bioconjugate Chemistry·Guangchang ZhouStephen J Kron
Oct 18, 2019·ACS Medicinal Chemistry Letters·Anjana SinhaSubhash C Sinha

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