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

Ding Wu, Michael R. Mand, Darren R. Veach, Laurie L. Parker, Bayard Clarkson, Stephen J. Kron

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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 specificity as a valuable means for efficient small molecule screening.

Original languageEnglish (US)
Pages (from-to)18-26
Number of pages9
JournalAnalytical Biochemistry
Volume375
Issue number1
DOIs
StatePublished - Apr 1 2008
Externally publishedYes

Bibliographical note

Funding Information:
We thank Ezra Abrams and Jelena Janjic for helpful advice and discussion and thank Wendy Stock for kindly providing imatinib. We also thank Won Jun Rhee and Stacey L. Kigar for synthesis of the peptides. Funding for this work was provided by National Institutes of Health grants R33 CA10323 and R01 HG003864. L.L.P. was supported by Kirchstein NRSA F32 CA117672. D.R.V. and B.C. were supported by the MeadWestvaco Corporation and Leukemia & Lymphoma Society grant 6034. S.J.K. was a Fletcher Scholar of the Cancer Research Foundation and a Leukemia & Lymphoma Society scholar.

Keywords

  • Imatinib mesylate (IM)
  • Kinase assay
  • Peptide substrate
  • Protein tyrosine kinase

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